Cell free compositions for cellular restoration and methods of making and using same

ABSTRACT

A pharmaceutical formulation comprising exosomes derived from mobilized stem cells, and one or more pharmaceutically acceptable carriers, adjuvants, or vehicles. A pharmaceutical formulation comprising a microvesicles derived from mobilized stem cells, and one or more pharmaceutically acceptable carriers, adjuvants, or vehicles. A pharmaceutical formulation comprising an exosome-derived and/or microvesicle derived molecule and one or more pharmaceutically acceptable carriers, adjuvants, or vehicles.

TECHNICAL FIELD

The present disclosure generally relates to compositions andmethodologies for the improvement and/or restoration of one or moreaspects of cellular function. More specifically this disclosure relatesto compositions comprising isolated restoring elements.

BACKGROUND

Aging is an important risk factor for most chronic diseases and is theprimary factor for the majority of morbidity and health careexpenditures in developed nations. Decreased cellular functionassociated with cellular senescence results in the disorders anddysfunctions typically associated with aging mammalian cells. A potentinducer of cellular senescence is (epi)genomic stress, which can resultfrom direct DNA damage, dysfunctional telomeres, disrupted chromatin, orstrong mitogenic signals. Additionally, cellular senescence can causechronic inflammation mediated, at least in part, bysenescence-associated secretory factors (SASF).

There exists an ongoing need for compositions and methods that improvecellular functions that have been negatively impacted due to one or moremechanisms associated with cellular senescence. Further, there exists anongoing need for compositions and methods to improve the cellular healthof a subject.

SUMMARY

Disclosed herein is a pharmaceutical formulation comprising exosomesderived from mobilized stem cells, and one or more pharmaceuticallyacceptable carriers, adjuvants, or vehicles.

Also disclosed herein is a pharmaceutical formulation comprising amicrovesicles derived from mobilized stem cells, and one or morepharmaceutically acceptable carriers, adjuvants, or vehicles.

Also disclosed herein is a pharmaceutical formulation comprising anexosome-derived and/or microvesicle derived molecule and one or morepharmaceutically acceptable carriers, adjuvants, or vehicles.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present disclosure and theadvantages thereof, reference is now made to the following briefdescription, taken in connection with the accompanying drawings anddetailed description, wherein like reference numerals represent likeparts.

FIG. 1 is a depiction of an embodiment for immunophenotyping a cellsample.

FIG. 2 is a depiction of an embodiment of a transwell co-cultureexperimental apparatus.

FIG. 3 is a plot of a gene expression analysis for donor cell samplesand receiver cell samples.

FIG. 4 is a plot of a protein expression analysis for donor cell samplesand receiver cell samples.

FIGS. 5A and 5B are a plot of a level of expression of the indicatedproteins for the donor cell samples and receiver cell samples.

FIG. 6 is a plot of the average telomere length for the donor cellsamples and receiver cell samples.

FIG. 7 is a plot of a gene expression analysis for baseline donor cellsamples and restored cell samples.

FIG. 8 is a plot of a protein expression analysis for baseline donorcell samples and restored cell samples.

FIG. 9A is a plot of a protein expression analysis for baseline donorcell samples and baseline receiver cell samples.

FIG. 9B is a plot of a protein expression analysis for baseline donorcell samples and baseline donor cell samples and restored cell samples.

FIG. 10A is a plot of a protein expression analysis for the baselinedonor cell sample and the baseline receiver cell sample R1.

FIG. 10B is a plot of a protein expression analysis for the baselinedonor cell sample and restored cell sample R1-D1.

FIG. 10C is a plot of a protein expression analysis for the baselinedonor cell sample and restored cell sample R1-D2.

FIG. 10D is a plot of a protein expression analysis for the baselinedonor cell sample and restored cell sample R1-D3.

FIG. 11 is a plot of a level of protein expression in restored cells inthe presence or absence of manumycin.

FIG. 12 is a plot of the telomere length for the restored cell samplefrom the donor cell sample-receiver cell sample pair R1-D1 in thepresence or absence of manumycin.

FIGS. 13 and 14 depict the results of the natural killer cell assay forthe samples from Example 6.

FIG. 15 depicts the results of the clonogenic assay for the samples fromExample 6.

FIGS. 16A-D depict the results of a flow cytometry assay for the samplesfrom Example 6.

DETAILED DESCRIPTION

Disclosed herein are compositions and methods for improving and/orrestoring one or more cellular functions. These cellular functions maybe directly or indirectly associated with promoting cellular health in asubject. Herein the term “promoting cellular health” refers toalterations in parameters of cellular function that result in aperceived and/or quantifiable improvement in the viability state ofcells and/or cell types. The viability state of a cell may be assessedusing any suitable metric to evaluate parameters such as, but notlimited to, cellular architecture, membrane organization and/orintegrity, dynamic protein assemblies, molecular organization, andcellular responses to external signals. The compositions and methodsdisclosed herein may improve the viability state of a cell as assessedby any suitable methodology. In an embodiment, a subject having improvedand/or restored cellular function via the compositions and/ormethodologies disclosed herein exhibits a perceived and/or quantifiableimprovement in one or more aspects of the subject's cellular and/orgeneral health.

The term “subject” as used herein, refers to an animal which is theobject of treatment, observation, or experiment. By way of example only,a subject may be, but is not limited to, a mammal including, but notlimited to, a human. The terms “treat,” “treating,” or “treatment,” asused herein, include alleviating, abating, or ameliorating a disease orcondition, or symptoms thereof; managing a disease or condition, orsymptoms thereof; preventing additional symptoms; ameliorating orpreventing the underlying metabolic causes of symptoms; inhibiting thedisease or condition, e.g., arresting the development of the disease orcondition; relieving the disease or condition; causing regression of thedisease or condition; relieving a condition caused by the disease orcondition; and/or stopping the symptoms of the disease or condition. Theterms “treat,” “treating,” or “treatment,” include, but are not limitedto, prophylactic and/or therapeutic treatments. “Prophylactic treatment”or “therapeutic treatment” refers to administration to the subject ofone or more of the disclosed compositions. If it is administered priorto clinical manifestation of an unwanted condition (e.g., medicalcondition, disease, disorder, dysfunction) then the treatment isprophylactic, i.e., it protects the subject against developing theunwanted condition, whereas if administered after manifestation of theunwanted condition, the treatment is therapeutic (i.e., it is intendedto diminish, ameliorate, or maintain the existing unwanted condition orside effects therefrom). It is contemplated that a subjectprophylactically treated for a medical condition with the compositionsand methodologies disclosed herein may nonetheless develop said medicalcondition; however, prophylactic treatment may be beneficial in terms oflessening the severity and/or duration of the medical condition.Treatment as used herein also encompasses any pharmaceutical ormedicinal use of the compositions herein.

In an embodiment, the subject is administered the compositions disclosedherein in a therapeutically effective amount sufficient for treating,preventing, and/or ameliorating one or more symptoms of a medicalcondition, disorder, disease, or dysfunction. Hereinafter, forsimplicity, the unwanted condition which has been used interchangeablywith the terms medical condition, disorder, disease, and dysfunction arecollectively referred to as the “medical condition.” As used herein,amelioration of the symptoms of the medical condition by administrationof a particular composition of the type disclosed herein refers to anylessening, whether lasting or transient, which can be attributed to orassociated with administration of compositions of the type disclosedherein. As used herein, a “therapeutically effective amount” means asufficient amount of the compositions disclosed herein to treat,prevent, and/or ameliorate one or more symptoms of the medicalcondition. It also may include a safe and tolerable amount of thecompositions disclosed herein, as based on industry and/or regulatorystandards. As will be understood by the ordinarily skilled artisan anamount that proves to be a “therapeutically effective amount” in a giveninstance, for a particular subject, may not be effective for 100% ofsubjects similarly treated for the medical condition underconsideration, even though such dosage is deemed a “therapeuticallyeffective amount” by ordinarily skilled practitioners. Thetherapeutically effective amount for a particular individual may varydepending on numerous factors such as the nature of the medicalcondition, severity of the medical condition, subject weight, subjectage, and the general health of the subject. It is contemplated that thetherapeutically effective amount may be optimized by one or morehealthcare professionals in consideration of the particular factorsaffecting a subject.

One or more compositions disclosed herein may comprise cells and/orcellular material obtained from a human subject. Herein the term“cellular material” refers to materials derived from, secreted by, andotherwise currently or previously associated with a cell.

In an embodiment, a method of the present disclosure comprises (i)obtaining a donor cell sample and a receiver cell sample; (ii) utilizingone or more analytical techniques to characterize the donor cell sampleand receiver cell sample; (iii) contacting one or more components of thedonor cell sample with the receiver cell sample to generate a restoredcell sample; (iv) utilizing one or more analytical techniques tocharacterize the restored cell sample; and (v) utilizing the restoredcell sample for treatment of a subject.

In an alternative embodiment, a method of the present disclosurecomprises (i) obtaining a donor cell sample and a receiver cell sample;(ii) contacting one or more components of the donor cell sample with thereceiver cell sample to generate a restored cell sample; and (iii)utilizing the restored cell sample for treatment of a subject.

In yet another embodiment, a method of the present disclosure comprises(i) obtaining a first cell sample from a first subject; (ii) obtaining asecond cell sample from a second subject; (iii) culturing the first cellsample in the presence of at least a portion of a culture media of thesecond cell sample for a time period ranging from about 24 hours toabout 6 weeks to produce a restoring composition; and (iv) contactingthe restoring composition with the second cell sample for a period oftime ranging from about 24 hours to about 6 weeks to produce a restoredcomposition.

In an embodiment, the donor cell sample is provided by a donor subjectwhile the receiver cell sample is provided by a receiver subject. Insome embodiments, the donor subject and receiver subject are the same.Alternatively, the donor subject and receiver subject are different. Inan embodiment, the donor subject is chosen such that the difference inthe age of the donor subject, designated x, and the age of the receiversubject, designated y, is greater than about 5 years, alternatively,greater than about 10 years, alternatively greater than about 15 years,alternatively greater than about 20 years, alternatively greater thanabout 25 years, or alternatively greater than about 30 years where y isgreater than x. In an embodiment, the donor subject is chosen such thatthe difference in the age of the donor subject, x, and the age of thereceiver subject, y, is from about 5 years to about 75 years,alternatively from about 10 years to about 60 years, alternatively fromabout 15 years to about 50 years, alternatively from about 20 years toabout 40 years, or alternatively from about 20 years to about 30 yearswhere y is greater than x.

In some embodiments, the difference in chronological age between thedonor subject and receiver subject is equal to or greater than about 16years, alternatively from about 16 years to about 80 years,alternatively from about 16 years to about 50 years, or alternativelyfrom about 16 years to about 30 years and x is greater than y. In yetanother embodiment, the difference in chronological age between thedonor subject and the receiver subject is less than about 365 days.

In an embodiment, the donor subject and receiver subject are related byconsanguinity. Alternatively, the donor subject and receiver subject arenot related. In an embodiment, the receiver subject has a medicalcondition that is absent from or undiagnosed in the donor subject. Ineither of the above disclosed embodiments, the donor subject and thereceiver subject are adults, i.e., have reached sexual maturity.Alternatively, in either of the above disclosed embodiments, the donorsubject has reached sexual maturity. Alternatively, in either of theabove disclosed embodiments, the receiver subject has reached sexualmaturity.

In an embodiment, the receiver subject is identified as having one ormore risk factors associated with the development of a medicalcondition. In yet another embodiment, the receiver subject has not beendiagnosed with a medical condition and/or has not been identified ashaving one or more risk factors associated with the development of amedical condition. It is contemplated that the methodologies disclosedherein may be employed in the treatment of subjects having a medicalcondition for which additional therapies have been previously or arecurrently being employed. It is further contemplated that in anembodiment, a receiver subject has undergone or is currently undergoingone or more therapies for medical conditions not associated with themedical condition for which the subject will be treated using thecompositions and methodologies disclosed herein. In an embodiment, thereceiver subject has one or more age-related medical conditions.

In an embodiment, the donor cell sample, receiver cell sample, or bothare obtained from a subject(s) who has undergone a Stage B preparation.In some embodiments, the donor cell sample, receiver cell sample, orboth are obtained from a subject(s) who has undergone a Stage Apreparation and a Stage B preparation.

In an embodiment, the donor cell sample, the receiver cell sample, orboth are obtained from a subject that has undergone a Stage Apreparation. Herein, a Stage A preparation of a subject comprises theutilization of methods and/or compositions to improve the subject'sgeneral health prior to obtaining a composition (i.e., donor cell sampleor receiver cell sample) from the subject.

A nonlimiting example of a methodology to improve the subject's generalhealth includes the administration of one or more metabolic mediators tothe subject. Herein, metabolic mediator refers to a substance which,when present in insufficient amounts in the subject, is detrimental tothe physiological and/or psychological state of the subject or whosepresence positively impacts the physiological and/or psychological stateof the subject. The subject may be administered a plurality of metabolicmediators prior to obtaining one or more compositions of the typedisclosed herein from the subject.

In an embodiment, the metabolic mediator comprises a nutraceutical.Herein, a nutraceutical refers to a material that may be derived from anatural source and that provides health benefits. A nonlimiting exampleof a nutraceutical suitable for use in the Stage A preparation of asubject is commercially available as EVERYCELL®, HEALTHYCELL, orHEALTHYCELL PLUS from Cell Health Institute. Additional compositionssuitable for use metabolic mediators in the present disclosure aredescribed in U.S. Pat. Nos. 8,747,915 and 8,974,839 entitled “DietarySupplement System for Multifunctional Anti-Aging Management and Methodof Use” which is incorporated by reference herein in its entirety.

Another example of a methodology suitable for use in Stage A preparationof a subject comprises the administration of one or more pulsedelectromagnetic fields (PEMF) to at least a portion of the subject'sbody prior to and/or concurrent with, obtaining a sample of the typedisclosed herein. PEMF may be used to enhance the homing, engraftment,and/or differentiation of the adult stem cells.

Stage A preparation of a subject may be carried out for some period oftime prior to, and/or concurrent with obtaining a cell sample of thetype disclosed herein from the subject. For example, Stage A preparationof a subject may comprise administration of a nutraceutical to thesubject at a particular dosage (e.g., 500 mg, twice daily) for a periodof time greater than about 48 hours prior to obtaining a cell sample ofthe type disclosed herein from the subject. Alternatively, thenutraceutical is administered for a time period of from about 48 hoursto about 1 year prior to obtaining a cell sample of the type disclosedherein from the subject, alternatively from about 1 week to about 9months, or alternatively from about 1 month to about 6 months. In someembodiments, the subject may be administered or may self-administer thenutraceutical for any period of time prior to, concurrent with, orsubsequent to the procurement of a cell sample.

In an embodiment, the donor cell sample, the receiver cell sample, orboth are obtained from a subject that has undergone a Stage Bpreparation. In an embodiment, during a Stage B preparation, the subject(donor and/or receiver) undergoes at least one process for mobilizingthe subject's stem cells. Herein “stem cells” are given their usualmeaning which generally refers to cells which are not terminallydifferentiated and are therefore able to produce cells of other types.Stem cells are typically divided into three types, including totipotent,pluripotent, and multipotent. “Totipotent stem cells” can grow anddifferentiate into any cell in the body, and thus can grow into anentire organism. These cells are not capable of self-renewal. Inmammals, the zygote and early embryonic cells are totipotent.“Pluripotent stem cells” are true stem cells, with the potential to makeany differentiated cell in the body, but cannot contribute to making theextraembryonic membranes (which are derived from the trophoblast).“Multipotent stem cells” are clonal cells that self-renew, as well asdifferentiate, to regenerate adult tissues. “Multipotent stem cells” arealso referred to as “unipotent” and can only become particular types ofcells, such as blood cells or bone cells.

In an embodiment, the donor and receiver cell samples comprise adultstem cells and/or adult stem cell material which refer to stem cells orstem cell material that are not embryonic in origin nor derived fromembryos or fetal tissue. In an alternative embodiment, the donor cellsample comprises adult stem cells and/or adult stem cell material whichrefer to stem cells or stem cell material that are not embryonic inorigin or derived from embryos or fetal tissue. In an embodiment, thedonor and receiver cell samples comprise stem cells and/or stem cellmaterial that are embryonic in origin and/or derived from embryos orfetal tissue. In an alternative embodiment, the donor cell samplecomprises stem cells and/or stem cell material that are embryonic inorigin and/or derived from embryos or fetal tissue.

In an embodiment, Stage B preparation comprises administering to asubject an effective amount of a mobilizer. Herein a “mobilizer” or a“mobilizer of hematopoietic stem cells or progenitor cells” (usedinterchangeably) refers to any substance, whether it is a small organicmolecule, synthetic or naturally derived, or a polypeptide, such as agrowth factor or colony-stimulating factor or an active fragment ormimic thereof, a nucleic acid, a carbohydrate, an antibody, or any otheragent that acts to enhance the migration of stem cells from the bonemarrow into the peripheral blood. Such a “mobilizer” may increase thenumber of stem cells (e.g., hematopoietic stem cells or hematopoieticprogenitor/precursor cells) in the peripheral blood, thus allowing for amore accessible source of stem cells for use in the methods disclosedherein. Any mobilizer suitable for increasing the number of stem cellsin the subject that are available to be harvested and is compatible withthe other aspects of this disclosure may be utilized. In an embodiment,the mobilizer is a cytokine such as granulocyte colony-stimulatingfactor (G-CSF). A commercial example of a mobilizer suitable for use inthe present disclosure is NEUPOGEN® (filgrastim) which is a prescriptionmedication used to treat neutropenia that is commercially available fromAmgen. Another example of a mobilizer suitable for use in the presentdisclosure is a recombinant methionyl human stem cell factor which iscommercially available as STEMGEN® from Amgen. Yet another example of amobilizer suitable for use in the present disclosure is plerixafor whichis an inhibitor of the CXCR4 chemokine receptor and blocks binding ofits cognate ligand, stromal cell-derived factor-1α (SCF-1α) and iscommercially available as MOZOBIL® from Genzyme.

An effective amount of a mobilizer may be determined by the ordinarilyskilled artisan consistent with best medical practices and taking intoaccount a variety of factors including, for example and withoutlimitation, the subject's general health and body mass.

As known to one of ordinary skill in the art, stem cells have beenidentified in various organs and tissues, including brain, bone marrow,peripheral blood, blood vessels, skeletal muscle, skin, teeth, heart,gut, liver, ovarian epithelium, and testis. It is contemplated thatutilization of Stage B preparation of a subject would be carried outwhen obtaining stem cells using bone marrow as the source. It is withinthe scope of this disclosure to conduct various embodiments of thepresent methods using cell samples comprising stem cells obtained fromany of the tissues known to be a source of stem cells. In suchembodiments, Stage B preparation of the subject may not be carried out.

In an embodiment, a donor subject, a receiver subject, or both undergoStage A preparation. In an embodiment, a donor subject, a receiversubject, or both undergo Stage B preparation. In an embodiment, a donorsubject, a receiver subject, or both do not undergo Stage A preparation.In an embodiment, a donor subject, a receiver subject, or both do notundergo Stage B preparation. In an embodiment, a donor subject and areceiver subject, or both undergo Stage A and Stage B preparation.

Subsequent to administration of the mobilizer, and after a suitable timeperiod has elapsed; a cell sample (e.g., donor cell sample or receivercell sample) may be harvested from a subject. The time period betweenadministration of the mobilizer to the subject and harvesting of thecell sample may be varied to meet one or more user and/or process goals.In an embodiment, the time period between administration of themobilizer and harvesting of the cell sample may range from about 24hours to about 10 days, alternatively from about 48 hours to about 7days, or alternatively from about 3 days to about 5 days.

In an embodiment, the cell sample is harvested from a subject using anysuitable methodology, for example, using an extracorporeal therapy suchas apheresis. Apheresis is a method used to collect only a specific partof the subject's blood. It works on the basis of centrifugation or rapidspinning of the blood. A pathway is established for the subject's bloodand allows for connection to the apheresis device. The instrument usessmall pumps to move blood and fluids through the system. One pump drawsblood out of one arm or side of the catheter and directs it to thecentrifuge where the blood is separated into red cell, white cell, andplasma layers. A portion of the white cell layer, which includes stemcells, and a small amount of plasma and red cells are diverted to acollection bag. The rest of the blood is returned to the subject in theother arm or the second side of the catheter. In such an embodiment, thecell sample is harvested using intravenous needles located in a vein ineach arm of a subject. Blood may be removed from a first vein, passedthrough an extracorporeal circuit that separates out the cell sample ofinterest and the remaining material may be returned to a second vein.

In an embodiment, the donor cell sample and/or receiver cell sample areharvested from the bone marrow directly. For example, the cell samplemay be harvested from the iliac crest of a subject. In such embodiments,bone marrow aspiration to obtain the cell sample may involve ahealthcare provider locating the posterior iliac crest of the subjectsubsequent to carrying out standard precautions such as skinsterilization and the administration of a local anesthetic. A suitableneedle with the stylet in place may be slowly advanced through the skinand subcutaneous tissue pointing towards the anterior superior iliacspine. Upon reaching the posterior iliac crest, the area may bepenetrated by the needle until an adequate depth is reached. Once theneedle is in place, the stylet may be removed, a syringe attached, andthe aspiration performed.

In an embodiment, a plurality of stem cell collections (e.g., bonemarrow aspirations) is carried out in order to obtain some user and/orprocess desired number of cells in the cell sample. For example, thenumber of cells collected may range from 1×10⁶-1.0×10⁹ cells/kg of thesubject weight, alternatively from about 2×10⁶-1.0×10⁸ cells/kg of thesubject weight, or alternatively from about 5×10⁶-1.0×10⁸ cells/kg ofthe subject weight. Cell samples harvested as disclosed herein may beutilized without further processing in the methodologies disclosedherein. Alternatively, cell samples harvested as disclosed herein may befurther processed using any methodology compatible with the compositionsand methodologies disclosed herein. Alternatively, cell samplesharvested as disclosed herein may be stored for some time period beforebeing utilized in the methodologies and therapies disclosed herein.Storage of the cell samples may involve, for example, cryogenicpreservation of the cell sample in a biocompatible solution to stabilizethe sample for the duration of storage. “Biocompatible solution” refersto solutions in which the cell sample (e.g., donor and/or receiver) aresuspended for use in the cellular restoration methodologies disclosedherein or for any other subsequent uses. Such biocompatible solutionsmay include saline and may further comprise other ingredients such aspreservatives, antimicrobials, and the like.

In an embodiment, cell samples harvested as disclosed herein are storedfor greater than about 24 hours prior to being utilized in themethodologies disclosed herein. Alternatively, the cell samplesharvested as disclosed herein are stored for a period of time rangingfrom about 1 hour to about 20 years prior to being utilized in themethodologies disclosed herein. Alternatively, storage of a cell sampleharvested as disclosed herein may be for a time period ranging fromabout 10 days to about 15 years, alternatively from about 30 days toabout 10 years, or alternatively from about 30 days to about 5 years.

As will be understood by the ordinarily skilled artisan, the donor cellsample and/or receiver cell sample, as harvested, comprise aheterogeneous cell population. An aspect of the methodologies disclosedherein comprises identifying and quantifying the types and amounts ofcells present in the donor cell sample and/or receiver cell sample. Anymethodology suitable for characterizing the number and types of cellspresent in the donor cell sample and/or receiver sample may be employed.In an embodiment, the donor cell sample and/or receiver cell sample arecharacterized by immunophenotyping. Herein, immunophenotyping refers tothe analysis of heterogeneous populations of cells for the purpose ofidentifying the presence and proportions of the various populations inthe sample. Antibodies are used to identify cells by detecting specificantigens (termed markers) expressed by these cells. In an embodiment,the donor cell sample and/or receiver sample are characterized byimmunophenotyping using techniques such as flow cytometry. Inalternative embodiments, characterizations of the various cell typespresent in a donor cell sample and/or receiver cell sample may becarried out using any suitable methodology such as reverse transcriptasepolymerase chain reaction (RT-PCR) or immunocytochemistry.

In an embodiment, the populations of cells or cell types present in thedonor cell sample and/or receiver cell sample are identified based onthe presence or absence or one or more cell surface markers. Anembodiment of a flow cytometry protocol for the identification of thedifferent populations of cells (e.g., cell types) in a donor cell sampleand/or receiver cell sample, 200, is presented in FIG. 1. Referring toFIG. 1, a cell sample (donor and/or receiver sample) 210 is subjected toflow cytometry. In an embodiment, the donor cell sample and/or receivercell sample 210 may be, at a first stage, sorted into hematopoieticcells 220 and non-hematopoietic cells 230 based on the presence orabsence of CD45. CD45, also known as leukocyte common antigen (LCA),T200, B220, Ly5, and protein tyrosine phosphatase receptor type C(PTPRC) is a transmembrane glycoprotein of theleukocyte-specific-receptor-like protein tyrosine phosphatase family. Itis expressed on all nucleated hematopoietic cells and can cover up to10% of the cell surface area. CD45 functions as a regulator of T-celland B-cell antigen receptor signaling and is a regulator of cell growthand cell differentiation.

In an embodiment, CD45− cells, identified as non-hematopoietic stemcells 230, may be further characterized on the basis of the presence orabsence of CD105. CD105, also known as endoglin, HHT1, ORW, and SH-1 isa type I membrane glycoprotein located on cell surfaces and is acomponent of the TGFβ receptor complex. CD105 may play a role inhematopoiesis and angiogenesis. In an embodiment, a cell population thatis both CD45− and CD105+, 240, is characterized as having bothmesenchymal stem cells and endothelial progenitor cells.

In an embodiment, a cell population that is identified to be both CD45−and CD105+, 240, may be further sorted into mesenchymal stem cells andendothelial progenitor cells. In an embodiment, the mesenchymal stemcells are identified as being CD45−, CD105+, CD29+ and CD44+, 250. CD29,also known as platelet GPIIa, integrin β1, and GP is an integrin unitassociated with very late antigen receptors and functions in celladhesion. CD44, also known as ECMRII, H-CAM, Pgp-1, HUTCH-1, Hermesantigen, phagocytic glycoprotein I, extracellular matrix receptor III,GP90 lymphocyte homing/adhesion receptor, and hyaluronate receptorfunctions in cell adhesion and migration. In an embodiment, endothelialprogenitor cells are identified as being CD45−, CD105+, and CD31+, 260.CD31, also known as PECAM-1, endoCAM, platelet endothelial cell adhesionmolecule, and PECA-1 is a protein that in humans is encoded by thePECAM1 gene found on chromosome 17. CD31 is thought to function in celladhesion, activation, and migration.

The method of the present disclosure may further comprise identifyingthe differing hematopoietic cell types present in the CD45+ cells, 220.In an embodiment, a population of the cells is identified as beingprimitive hematopoietic stem cells, 270, on the basis of being CD45+,CD34+ and CD38−. In an embodiment, a population of the cells isidentified as being hematopoietic progenitor cells on the basis of beingCD45+, CD34+ and CD38+, 280. CD34 also known as gp105-120 andhematopoietic progenitor cell antigen (HPCA-1) is a member of the familyof single-pass transmembrane sialomucin proteins that are expressed onearly hematopoietic and vascular tissues. CD34 is thought to function incell adhesion. CD38, also known as ADP-ribosyl cyclase, T10, and cyclicADP-ribose hydrolase 1 is a multifunctional ectonucleotidase encoded bythe CD38 gene which is located on chromosome 4. In an embodiment, atleast a portion of the cell population are CD45+ and CD34−, 290, and areidentified as differentiated hematopoietic cells. In such an embodiment,the differentiated hematopoietic cells, 290, may be further defined asbeing T-lymphocytes, 300, or Natural Killer cells, 310. T-lymphocytescan be characterized as being CD45+, CD34−, and CD3+. CD3, also known asT3, is a protein complex and plays a role in cell adhesion betweenT-cells and other cell types. Natural Killer cells can be characterizedas being CD45+, CD34−, and CD56+. CD56 also known as Leu-19, NKH-1, andneural cell adhesion molecule (NCAM) is a hemophilic bindingglycoprotein that may function in cell-cell adhesion, neurite outgrowth,synaptic plasticity, and learning and memory.

In an embodiment, the donor cell sample and/or receiver sample may becharacterized using the methodologies disclosed herein. Suchcharacterizations may result in the identification of cell populationsin the donor cell sample and/or receiver cell sample that includewithout limitation, non-hematopoietic cells, mesenchymal stem cells,endothelial progenitor cells, hematopoietic cells, primitivehematopoietic stem cells, hematopoietic progenitor cells, differentiatedhematopoietic cells, T-lymphocytes, natural killer cells, orcombinations thereof. It is contemplated that the surface markersdescribed herein represent one methodology for the identification ofcell populations present within the donor cell sample and/or receivercell sample. As will be understood by the ordinarily skilled artisan,numerous markers and combination of markers other than those disclosedherein may be utilized to identify and characterize the cell populationspresent within the donor cell sample and/or receiver cell sample.Further, the identification of the various cell populations present inthe donor cell sample and/or receiver cell sample may be carried out tothe extent described herein, may include determination of the presenceor absence of additional surface markers, may utilize fewer markers thandisclosed herein, or may be carried out to a lesser extent such thatfewer populations of cells within the donor cell sample and/or receivercell sample are identified. In an embodiment, a method comprisesexcluding the identification of the different populations of cellspresent in a donor cell sample and/or receiver cell sample.

In an embodiment, a donor cell sample and/or receiver cell sample isobtained from a subject having undergone a Stage B preparation. In suchembodiments, the donor cell sample and/or receiver cell sample may befurther characterized based on the number of senescent cells andnon-senescent cells present in the cell sample. Herein, non-senescentcells refer to the cells that retain the ability to divide many timesover without showing replicative senescence. Herein senescent cellsrefer to cells having a long-term loss of proliferative capacity despitecontinued viability and metabolic activity.

Senescent cells may be identified using a variety of metrics thatinclude for example loss of proliferation, morphological changes,decreased telomere lengths, increased S-β-GAL activity, the productionof senescence-associated heterochromatic foci (SAHF), increasedproduction of senescence-associated secretory factors (SASF), increasedproduction of reactive oxygen species (ROS), increased DNA damage,decreased chaperone-mediated autophagy, or combinations thereof. It iscontemplated that changes in the various metrics described are assessedrelative to comparable cell types established to be non-senescent cells.Alternatively, the characteristics of the cell sample may be compared toliterature values established for the analyzed metric in a correspondingnon-senescent cell.

Non-senescent cells may characterized by the length of their telomeresand of the level of telomerase activity present in the cell. By way of anon-limiting example, non-senescent cells present in the donor cellsample may be characterized by telomere lengths greater than or equal toabout 4 kilobases, alternatively 4.5 kilobases, or alternatively 5kilobases. It will be understood by the ordinarily skilled artisan thatteleomere lengths indicative of non-senescent cells may vary dependingon the cell type. Consequently, for a particular cell type, the telomerelength characteristic of a non-senescent cell may be determined byroutine experimentation.

In an embodiment, Stage B preparation of the subject from which thedonor cell sample and/or receiver cell sample is harvested results inthe preferential mobilization of non-senescent cells. The result of thepreferential mobilization of non-senescent cells may be a donor cellsample and/or receiver cell sample comprising greater than 90%non-senescent cells, alternatively greater than 91% non-senescent cells,alternatively greater than 92% non-senescent cells, alternativelygreater than 93% non-senescent cells, alternatively greater than 94%non-senescent cells, alternatively greater than 95% non-senescent cells,alternatively greater than 96% non-senescent cells, alternativelygreater than 97% non-senescent cells, alternatively greater than 98%non-senescent cells, or alternatively greater than 99% non-senescentcells. The percentage of non-senescent cells is based on the totalnumber of cells present in the sample. In an embodiment, the donor cellsample and/or receiver cell sample comprise from about 90% non-senescentcells to about 99% non-senescent cells based on the total number ofcells present in the sample.

In some embodiments, the non-senescent cells present in the donor cellsample and/or receiver cell sample may be identified using any suitablemethodology. In such embodiments, the non-senescent cells may beseparated from the senescent cells using any suitable process compatiblewith the present disclosure to result in a donor cell sample and/orreceiver cell sample that comprises, consists essentially of, orconsists of non-senescent cells. It is contemplated that suchmethodologies may be extended to further define a population ofnon-senescent cells having the presence or absence of particular cellsurface markers and result in a donor cell sample and/or receiver cellsample comprising, consisting essentially of, or consisting ofnon-senescent cells of a particular type (e.g., non-senescentmesenchymal stem cells, non-senescent natural killer cells).

In an embodiment, the donor cell sample and/or receiver cell sample maybe analyzed for the extent of expression of one or more genes and/orproteins associated with cellular senescence. Such analyses may becarried out using a restoration biomarker protein panel (RBPP) and/orrestoration biomarker gene expression panel (RBGEP) of the typesdisclosed herein.

In an embodiment, the RBPP comprises a plurality of antibody probes forfactors linked to cellular aging and senescence. For example, the RBPPmay comprise greater than 5 antibody probes, alternatively greater than10 antibody probes, or alternatively greater than 20 antibody probes. Inan embodiment the RBPP comprises from 10 to 15 antibody probes. Anexample of a RBPP suitable for use in this disclosure is a protein arraypanel designated RBPP-X1 comprising antibody probes to the proteinslisted in Table 1:

TABLE 1 Name Also Known As Designated granulocyte-colony stimulatingcolony-stimulating factor 3 G-CSF factor chemokine ligand 26 eotaxin-3,macrophage inflammatory protein 4-alpha, CCL26 thymic stroma chemokine,and IMAC hepatocyte growth factor hepatocyte scatter factor (HSF), HGFinsulin-like growth factor binding placental protein 12 (PP12) IGFBP-1protein 1 insulin-like growth factor binding IGFBP-4 protein 4insulin-like growth factor binding IGFBP-6 protein 6 insulin-like growthfactor beta catabolin IL-β macrophage inflammatory protein chemokineligand 20, liver activation regulated MCP-3α 3 (MIP3A) chemokine (LARC)stem cell factor KIT-ligand, KL, steel factor SCF thymus and activationregulated chemokine ligand 17 (CCL17), TARC chemokine transforminggrowth factor beta 1 TGF-β1 tumor necrosis factor receptor sTNFR1superfamily member 1A vascular endothelial growth factor VEGF

In an embodiment, the RBGEP may comprise greater than 5 gene probes,alternatively greater than 10 gene probes, or alternatively, greaterthan 20 gene probes. In an embodiment, the RBGEP comprises from 10 to 15gene probes. In some embodiments, the RBGEP comprises gene probes forfactors linked to the regulation of cell cycle or the p53 pathway suchas IFBP3, CSC25C, ABL1, CDKN2B, ALDH1A3, SIRT1, ING1, CITED2, andCDKN1C. The RBGEP may further comprise gene probes for factorsassociated with regulation of inflammatory processes such as CDKN1A,IRF3, EGR1, IFNG, CDKN1B, NFKB1, SERPING2, IGFBP7, and IRF7. The RBGEPmay further comprise gene probes for factors associated with regulationof DNA damage related-processes such as PCNA, TERT, and TP53BP1. TheRBGEP may further comprise gene probes for factors associated withoxidative stress such as PRKCD, SOD1, and NOX4. The RBGEP may furthercomprise gene probes for factors associated with cellular senescencesuch as CDKN2A, CDK6, TWIST, ATM, CCND1, ETS2, RBL2, BMI1, and ETS1. TheRBGEP may further comprise gene probes for factors associated with theMAPK pathway such as HRAS and MAP2K3. The RBGEP may further comprisegene probes for factors associated with cytoskeletal function such asVIM, PIK3CA, and THBS1. The RBGEP may further comprise gene probes forfactors associated with the p16 effector pathway such as TBX3 and TBX2.The RBGEP may further comprise gene probes for factors associated withinsulin signaling such as IGFBP5. The RBGEP may further comprise geneprobes for factors associated with cell adhesion such as CDL3A1, CD44,TGFB1A, CDL1A1 and TGFB1. The RBGEP may further comprise gene probes forfactors associated with the p53 effector pathway such as E2F1 and MYC.An example of a RBGEP suitable for use in this disclosure, designatedRBGEP-X1, is a gene panel comprising cDNA to the proteins listed inTable 2:

TABLE 2 Gene Protein Encoded IGFBP3 insulin-like growth factor bindingprotein 3 HRAS Transforming protein p21 PRKCD protein kinase C deltaAKT1 alpha serine/threonine protein kinase CHEK2 checkpoint kinase 2MAPK14 mitogen-activated protein kinase 14 IGF1 insulin-like growthfactor TWIST1 Twist-related protein 1 CDC25C M-phase inducer phosphatase3 CCNA2 cyclin-A2 CDK5 cell-division protein kinase 6 CCNE1G1/S-specific cyclin E1 CHEK1 checkpoint kinase 1

In an embodiment, at least a portion of the donor cell sample and/orreceiver cell sample are subjected to protein array analyses utilizingthe RBPP-X1 array, gene expression analysis using the RBGEP-X1 array, orboth. In alternative embodiments, at least a portion of the donor cellsample and/or receiver cell sample are subjected to protein arrayanalyses, gene expression analyses or both utilizing any suitableprotein and/or gene array.

In an embodiment, the donor cell sample, receiver cell sample, or bothare subjected to at least one analytical technique to characterize thequality of the cell sample. Herein, the “quality” of the cell samplerefers to factors used to characterize the cellular health of the sampleand includes parameters such as the number and types of cells present inthe sample; the ratio of senescent to non-senescent cells in the sample;the extent of expression of a group of genetic and/or proteinbiomarkers; the average telomere length of the cells in the sample; andthe status of the innate immune function of the cells in the sample.Telomere length may be determined using any suitable methodology, forexample, terminal restriction fragment (TRF) analysis. Innate immunefunction may be evaluated using any suitable methodology such as the⁵¹Cr cytotoxicity release natural killer cell assay. The donor cellsample quality may be an assessment of the ability of the cells in thesample to improve and/or restore one or more cellular functions of thecells in the receiver cell sample. The receiver cell sample quality maybe an assessment of the ability of the cells in the sample to exhibitimprovement and/or the restoration of one or more cellular functionswhen subjected to the compositions and methodologies disclosed herein.

The donor cell sample quality may be assigned a numerical value thatranges from 1 to 10 wherein a sample displaying positive characteristicsfor use in the improvement and/or restoration of cellular function of areceiver cell sample has a value of 10, and a sample exhibiting thefewest characteristics associated with the ability to improve/restorecellular function of a receiver cell sample has a value of 1. Forexample, each of the following factors may weigh positively incharacterization of the quality of a donor cell sample; relatively longtelomere length; high level of expression of cell viability-promotinggenes and/or proteins; the presence of greater than about 90%non-senescent cells; and high levels of innate immune function. Donorcell samples displaying these characteristics may be given a samplequality value of 10.

The receiver cell sample quality may be assigned a numerical value thatranges from 1 to 10 wherein a sample having restorable or improvablecellular function has a value of 10, and a sample whose cellularfunction cannot be significantly improved and/or restored has a valueof 1. For example, each of the following factors may weigh positively incharacterization of the quality of a receiver cell sample; relativelylong telomere length; moderate level of expression ofsenescence-promoting genes and/or proteins; and the presence of greaterthan about 90% non-senescent cells. Receiver cell samples displayingthese characteristics may be given a sample quality value of 10.

Utilizing the quality metrics disclosed herein (e.g., telomere length,percentage of non-senescent cells), an aspect of the present disclosurecomprises evaluating the quality of the donor cell sample and receivercell sample and identifying samples suitable for use in the disclosedmethodologies. For example, a receiver cell sample having a qualityvalue of less than 3 may be deemed unsuitable for use in the presentlydisclosed methodologies. Similarly, a donor cell sample having a qualityvalue of less than 3 may be deemed unsuitable for use in the presentmethodologies. In some embodiments, the a donor cell sample having aquality value of equal to or greater than 7 may be used in themethodologies disclosed herein with a receiver cell sample having aquality value of equal to or greater than 7. It is to be understood thatthe quality values may be assigned based on any number of metrics usedto assess the quality of a donor cell sample and/or receiver cellsample. Consequently, based on the parameters used to make theassignment of a quality value, the characteristics associated with aparticular quality value may differ.

In some embodiments, the donor cell sample and/or receiver cell samplehaving been subjected to one or more of the qualitative and quantitativecharacterizations described herein are further processed to provide someuser and/or process desired sample containing a predetermined type andnumber of cells. The present disclosure contemplates the utilization ofsuch characterized samples. For example, the characterized samples maybe a component of a pharmaceutical formulation that is administered to asubject to ameliorate one or more medical conditions.

Alternatively, the donor cell sample and receiver cell sample may beutilized in the restoration methodologies disclosed herein.

In an embodiment, a method of cellular restoration comprises contactingthe soluble factors and/or particles present in the media of a cultureddonor cell sample with the receiver cell sample. For example, the donorcell sample may be cultured in appropriate media for a time periodranging from about 24 hours to about 6 weeks, alternatively, from about1 week to about 5 weeks or alternatively, from about 2 weeks to about 4weeks. Herein, the culture media, also known as the growth media, refersto a liquid or gel containing the appropriate nutrients to support thegrowth of cells. Suitable culture media may be chosen by the ordinarilyskilled artisan with the benefits of the present disclosure. The culturemedia may then be removed from the donor cell sample using any suitablemethodology (e.g., filtration, centrifugation) and the cell-free mediathen contacted with the receiver cell sample.

In an alternative embodiment, the method of cellular restorationcomprises establishing a transwell culture of both the donor cell sampleand receiver cell sample. Referring to FIG. 2, the transwell culture 400may comprise an insert 410 having at least one permeable surface thatallows the donor cells to uptake and secrete molecules on the basaland/or apical surfaces of the transwell. The transwell insert 410 may becomprised of any material compatible with the compositions andmethodologies disclosed herein such as, for example, polyethyleneterephthalate or polycarbonate. In an embodiment, the transwell insert410 comprises a permeable membrane with a pore size ranging from 0.4 μmto 3.0 μm, alternatively from 0.4 μm to 2.0 μm, or alternatively from0.4 μm to 1.0 μm. The transwell insert may have a pore size that allowsfor the passage of soluble factors and/or particles secreted or releasedfrom the donor cell sample to the lower compartment of the transwellwhere these materials contact the receiver cell sample. At least aportion of the donor cell sample 420 may be applied to the transwellinsert 410 while the receiver cell sample 430 is positioned within thelower compartment of the transwell culture with an appropriate amount ofculture media. The donor cell sample and receiver cell sample may becultured in the transwell for a time period of from 24 hours to 6 weeks,alternatively, from 1 week to 5 weeks, or alternatively, from 2 weeks to4 weeks. Soluble factors and/or particles of the appropriate size 440are allowed to pass through the permeable membrane and contact thereceiver cell sample 430 in the lower chamber of the transwell.

In an embodiment, the donor cell sample and receiver cell sample arerecovered separately from the transwell culture. The donor cell sample,as recovered from the transwell culture, is hereinafter termed thealtered donor cell sample (ADCS). The receiver cell sample as recoveredfrom the transwell culture is termed the restored composition (RC). Inan embodiment, the ADCS and/or RC may be further characterized using anyof the methodologies disclosed herein. In some embodiments, at least aportion of the ADCS and/or RC are further processed, for example, thesamples may be prepared for cryopreservation. In yet another embodiment,at least a portion of the RC is utilized to treat a subject.

Herein the RC refers to the cellular material subsequent to culturingwith the soluble factors of the donor cell sample for the time periodsdisclosed herein. The RC is characterized by improvement in one or moreof the following metrics when compared to the receiver cell sample;innate immune function, morphology, colony-forming ability, reducedexpression of senescence-promoting factors; increased expression ofcell-viability promoting factors, and the like. In an embodiment, the RCis characterized by the presence of cells having gene expression andprotein expression patterns for cellular senescence associated agents(e.g., CDKN2A, CDK6, TWIST, ATM, CCND1, ETS2, RBL2, BMI1, and ETS1) thatare quantitatively more similar to those of the donor cell sample thanthe receiver cell sample.

In one embodiment, a methodology disclosed herein comprises thepreparation of a RC. The RC is derived from the receiver cell samplethat is subjected to the methodologies disclosed herein, specifically bythe restoration of at least a portion of the receiver cell sample.Herein, “restoration” refers to modification of a cell (e.g., stem cell)such that expression of one or more senescence-promoting agents isreduced and/or expression of one or more cell viability/cellfunction-promoting agents is increased. Without wishing to be limited bytheory, the methodologies and compositions disclosed herein may resultin the epigenetic modification of one or more cell types that results inat least one characteristic associated with improved cellular functionwhen compared to an otherwise similar cell type not subjected to thecompositions and methods disclosed herein. Herein, “epigenetic” refersto the heritable changes in gene activity and expression that occurswithout alternation in DNA sequence. Nonlimiting examples of epigeneticmodifications include posttranslational modifications such as DNAmethylation, chromatin remodeling, and histone modification.

The RC comprises cells that may exhibit alterations in parameters ofcellular and/or organismal physiology that result in a perceived and/orquantifiable improvement in the functional state of receiver cellsand/or cell types, wherein perceived improvement is defined as semblanceto the functional state of the donor cells and/or cell types. It is tobe understood that the restored composition comprises cells and isderived from a corresponding receiver cell sample.

In an embodiment, a RC is characterized by the maintenance of theviability state of the cells and/or cell types in the composition asquantified, for example, by a cell vitality assay.

In an alternate embodiment, a RC is characterized by an increase in theviability state of the cells and/or cell types as quantified, forexample, by a cell viability assay.

In an embodiment, a RC is characterized by a lack of change in thepercentage of hematopoietic stem cells, hematopoietic progenitor cells,mesenchymal stem cells and endothelial progenitor cells, herein termedthe “stem cell pool”, compared to the receiver cell sample.

In an alternate embodiment, a RC is characterized by an increase in thestem cell pool in comparison to the receiver cell sample.

In an embodiment, a RC is characterized by cells that exhibit animprovement in cellular immune function in comparison to thecorresponding receiver cell sample as quantified, for example, bynatural killer cell cytotoxicity assay.

In an embodiment, a RC is characterized by cells that exhibit animprovement in cellular hematopoietic function as quantified, forexample, by hematopoietic stem cell clonogenic assay in comparison tothe corresponding receiver cell sample.

In an alternate embodiment, a RC is characterized by cells that exhibitan improvement in systematic hematopoietic and immune function of thesubject when compared to the corresponding receiver cell sample asquantified, for example, by increased lymphopoiesis, increased ratio ofCD4 to CD8 positive T cells, and/or improved immune surveillance.Improved immune surveillance can be determined by decreased incidencesof microbial infection and tumor formation in the subject. Improvedimmune surveillance can also be measured by decreased rate of cancerincidence in the subject. Decreased rates of cancer incidence shallthereby confer to the subject an increased likelihood of prolongedorganismal survival.

In an embodiment, a RC is characterized by cells that exhibit aminimization of replicative stress as determined, for example, bytelomere length and/or telomerase activity when compared to thecorresponding receiver cell sample.

In an embodiment, a RC is characterized by cells that exhibit adecreased expression of senescence-related genes when compared to thecorresponding receiver cell sample, wherein senescence-related genes aredefined, for example, as the RBGEP, by quantitative polymerase chainreaction.

In an embodiment, a RC is characterized by cells that exhibit adecreased expression of senescence-associated secretory factors whencompared to the corresponding receiver cell sample, whereinsenescence-associated secretory factors are exemplified in Table 1.

In an embodiment, a RC is characterized by cells that exhibitalterations in the epigenetic signature of the cells when compared tothe corresponding receiver cell sample, wherein epigenetic signature isdetermined, for example, by chromatin immunoprecipitation sequencing(ChIP-Seq).

In an embodiment, a RC is characterized by cells that exhibit anincrease in the rate of proteostasis when compared to the correspondingreceiver cell sample which may be quantified, for example, by a Cyto-ID®Autogphagy Detection Kit.

In an embodiment, a RC is characterized by cells that exhibit a decreasein cellular oxidative stress when compared to the corresponding receivercell sample, as quantified for example by the MitoSOX™ Red mitochondrialsuperoxide indicator kit.

In an embodiment, a RC is characterized by cells that exhibit a decreasein cellular senescence when compared to the corresponding receiver cellsample, as quantified, for example, by the Fluorometric QuantitativeCellular Senescence β-Gal Assay Kit.

In an embodiment, a RC is characterized by cells that exhibit themaintenance of mesenchymal stem cell function when compared to thecorresponding receiver cell sample, wherein mesenchymal stem cellfunction is quantified, for example, by the colony formingunit-fibroblast (CFU-F) assay and/or ability to undergo lineage-specificdifferentiation into adipogenic, osteogenic and chondrogenic lineages.

In an alternate embodiment, a RC is characterized by cells that exhibitan increased mesenchymal stem cell function when compared to thecorresponding receiver cell sample.

In an embodiment, a RC is characterized by cells that exhibitmaintenance of endothelial progenitor function when compared to thecorresponding receiver cell sample wherein endothelial progenitorfunction is quantified, for example, by tube formation assay.

In an alternate embodiment, a RC is characterized by cells that exhibitan increased endothelial progenitor function when compared to thecorresponding receiver cell sample.

Herein, cellular restoration occurs following contact of the receivercell sample with soluble factors and/or particles present in the donorcell sample (e.g., materials that pass through the permeable transwellinsert). Consequently, the method of restoration comprises contact ofthe cell-free soluble factors and/or particles present in the media of adonor cell sample with a receiver cell sample. In some embodiments, thedonor cell sample is cultured in a suitable media and the media may thenbe separated from the donor cells to form a cell-free media which isutilized in the restoration of a receiver cell sample. Without wishingto be limited by theory, the soluble factors and/or particles present inthe donor cell sample media that pass through the permeable transwellinsert may include paracrine factors, microvesicles, exosomes, cellularfragments, and the like Herein paracrine factors refer to signalingmolecules which are secreted into the immediate extracellularenvironment and diffuse over a short distance to a target cell.Microvesicles generally refer to small (e.g., 50 nm to 100 nm) fragmentsof plasma membrane thought to be shed by a variety of cell types.Exosomes generally refer to secreted extracellular vesicles that maycontain biomolecules such as proteins, lipids, and RNA and function incellular signaling. In an embodiment, the soluble factors and/orparticles of the donor cell sample that contact the receiver cell samplecomprise extracellular vesicles. Exosomes and microvesicles belong to abroader group of extracellular vesicles (EVs) that represent animportant mode of intercellular communication by serving as vehicles fortransfer between cells of membrane and cytosolic proteins, lipids, andRNA.

In an embodiment, the quality of the restoring composition can beadjusted by the presence of one or more materials that regulate therelease of EVs. For example, the release of one or more EVs may beinhibited by the addition of small molecule inhibitors such asmannumycin A. Alternatively, the release of EVs may be promoted, forexample, by activation of purinergic receptors with ATP, activation bylipopolysaccharides, plasma membrane depolarization, or increasingintracellular Ca²⁺ concentrations.

It is a contemplated aspect of the present disclosure that cell-freemedia generated by culturing of the donor cell sample in a suitablemedia followed by removal of the cells and herein designated therestoring composition, may be further processed to separate individualconstituents or groups of constituents based on like characteristicsusing any suitable methodology (e.g., ethanol precipitation,centrifugation gradients). In an embodiment, the individual constituentsof the restoring composition may be analyzed for their ability to affectrestoration of a receiver cell sample of the type disclosed herein. Thepresent disclosure further contemplates utilization of one or moreisolated constituents, or isolated groups of constituents, in themethodologies for cellular restoration. In an embodiment, cellularrestoration of a receiver cell sample of the type disclosed herein iscarried out utilizing an EV (e.g., exosome/microvesicle) isolated from aculture media of a donor cell sample. In another embodiment, cellularrestoration of a receiver cell sample of the type disclosed herein iscarried out utilizing cellular fragments isolated from a culture mediaof a donor cell sample.

In an embodiment, the RC may be formulated for administration to asubject in need thereof. In an embodiment, the subject is the receiversubject. For example, the RC may be a component of a formulation that isadministered to the receiver subject to improve the receiver subject'sgeneral health. Such improvements may be identified by quantitativeevaluation of one or more physiological or psychological parameters ofthe subject. In the alternative, such improvements may be identified bythe qualitative evaluations of one or more physiological orpsychological parameters of the subject. In an embodiment, the receiversubject is prophylactically administered the RC.

The RC may be administered to a subject (e.g., receiver subject) via anysuitable methodology. In some embodiments, the methodologies disclosedherein comprise systemic administration of the RC to the subject. Forexample, the RC may be administered systemically in dosage unitformulations containing conventional nontoxic pharmaceuticallyacceptable carriers, adjuvants, and vehicles as desired. In a specificembodiment, administration of the RC may be by intravenous injection,endobronchial adminstration, intraaterial injection, intramuscularinjection, intracardiac injection, subcutaneous injection,intraperitoneal injection, intraperitoneal infusion, transdermaldiffusion, transmucosal diffusion, intracranial, intrathecal, orcombinations thereof. A means of administering the RC may include, butis not limited to, infusion. Systemically may also include, for example,by a pump, by an intravenous line, or by bolus injection. Bolusinjection can include subcutaneous, intramuscular, or intraperitonealroutes.

The phrases “systemic administration” or “administered systemically,” asused herein, mean the administration of a compound(s) of the disclosure,composition, drug, or other material such that it enters the subject'ssystem and, thus, is subject to metabolism and other like processes, forexample, subcutaneous administration.

In other embodiments, the RC is locally administered by means such as,but not limited to, injection, implantation, grafting, or epicutaneous.For example, the RC may be administered proximal to a wound site on thesubject and functions to ameliorate the symptoms associated with thewound or increase the rate of wound-healing. In yet other embodiments,the RC may be administered at or proximal to the site utilized toharvest the receiver cell sample from the subject. Administration of theRC may be conducted in any manner compatible with the compositionsdisclosed herein and to meet one or more user and/or process goals.

In an alternative embodiment, the subject (e.g., receiver subject) isadministered the RC as a component of a therapeutic procedure designedto ameliorate the effects of a medical condition. In such embodiments,the RC, present in a therapeutically effective amount, may function asan active agent in a pharmaceutical composition. Such pharmaceuticalcompositions comprising the RC are hereinafter termed cellularmaterial-based pharmaceutical compositions or CMBPC. Additional activeagents may be present in the CMBPC as considered beneficial for thetreatment of the medical condition. Examples of additional active agentsinclude but are not limited to: (a) antimicrobials, (b) steroids (e.g.,hydrocortisone, triamcinolone); (c) pain medications (e.g., aspirin, anNSAID, and a local anesthetic); (d) anti-inflammatory agents; (e) growthfactors; (f) cytokines; (g) hormones; and (h) combinations thereof. Suchadditional active agents may also be present in a therapeuticallyeffective amount.

Examples of additional active agents for inclusion in the CMBPCpharmaceutical or drug include, but are not limited to, anesthetics,hypnotics, sedatives and sleep inducers, antipsychotics,antidepressants, antiallergics, antianginals, antiarthritics,antiasthmatics, antidiabetics, antidiarrheal drugs, anticonvulsants,antigout drugs, antihistamines, antipruritics, emetics, antiemetics,antispasmodics, appetite suppressants, neuroactive substances,neurotransmitter agonists, antagonists, receptor blockers and reuptakemodulators, beta-adrenergic blockers, calcium channel blockers,disulfuram and disulfuram-like drugs, muscle relaxants, analgesics,antipyretics, stimulants, anticholinesterase agents, parasympathomimeticagents, hormones, anticoagulants, antithrombotics, thrombolytics,immunoglobulins, immunosuppressants, hormone agonists/antagonists,vitamins, antimicrobial agents, antineoplastics, antacids, digestants,laxatives, cathartics, antiseptics, diuretics, disinfectants,fungicides, ectoparasiticides, antiparasitics, heavy metals, heavy metalantagonists, chelating agents, gases and vapors, alkaloids, salts, ions,autacoids, digitalis, cardiac glycosides, antiarrhythmics,antihypertensives, vasodilators, vasoconstrictors, antimuscarinics,ganglionic stimulating agents, ganglionic blocking agents, neuromuscularblocking agents, adrenergic nerve inhibitors, anti-oxidants, vitamins,cosmetics, anti-inflammatories, wound care products, antithrombogenicagents, antitumoral agents, antiangiogenic agents, anesthetics,antigenic agents, wound healing agents, plant extracts, growth factors,emollients, humectants, rejection/anti-rejection drugs, spermicides,conditioners, antibacterial agents, antifungal agents, antiviral agents,antibiotics, tranquilizers, cholesterol-reducing drugs, antitussives,histamine-blocking drugs, and monoamine oxidase inhibitor.

Specific compounds suitable for use in the CMBPC include silversulfadiazine, Nystatin, Nystatin/triamcinolone, Bacitracin,nitrofurazone, nitrofurantoin, a polymyxin (e.g., Colistin, Surfactin,Polymyxin E, and Polymyxin B), doxycycline, antimicrobial peptides(e.g., natural and synthetic origin), NEOSPORIN® (i.e., Bacitracin,Polymyxin B, and Neomycin), POLYSPORIN® (i.e., Bacitracin and PolymyxinB). Additional antimicrobials include topical antimicrobials (i.e.,antiseptics), examples of which include silver salts, iodine,benzalkonium chloride, alcohol, hydrogen peroxide, chlorhexidine,acetaminophen; Alfentanil Hydrochloride; Aminobenzoate Potassium;Aminobenzoate Sodium; Anidoxime; Anileridine; Anileridine Hydrochloride;Anilopam Hydrochloride; Anirolac; Antipyrine; Aspirin; Benoxaprofen;Benzydamine Hydrochloride; Bicifadine Hydrochloride; BrifentanilHydrochloride; Bromadoline Maleate; Bromfenac Sodium; BuprenorphineHydrochloride; Butacetin; Butixirate; Butorphanol; Butorphanol Tartrate;Carbamazepine; Carbaspirin Calcium; Carbiphene Hydrochloride;Carfentanil Citrate; Ciprefadol Succinate; Ciramadol; CiramadolHydrochloride; Clonixeril; Clonixin; Codeine; Codeine Phosphate; CodeineSulfate; Conorphone Hydrochloride; Cyclazocine; DexoxadrolHydrochloride; Dexpemedolac; Dezocine; Diflunisal; DihydrocodeineBitartrate; Dimefadane; Dipyrone; Doxpicomine Hydrochloride; Drinidene;Enadoline Hydrochloride; Epirizole; Ergotamine Tartrate; EthoxazeneHydrochloride; Etofenamate; Eugenol; Fenoprofen; Fenoprofen Calcium;Fentanyl Citrate; Floctafenine; Flufenisal; Flunixin; FlunixinMeglumine; Flupirtine Maleate; Fluproquazone; Fluradoline Hydrochloride;Flurbiprofen; Hydromorphone Hydrochloride; Ibufenac; Indoprofen;Ketazocine; Ketorfanol; Ketorolac Tromethamine; Letimide Hydrochloride;Levomethadyl Acetate; Levomethadyl Acetate Hydrochloride; LevonantradolHydrochloride; Levorphanol Tartrate; Lofemizole Hydrochloride;Lofentanil Oxalate; Lorcinadol; Lomoxicam; Magnesium Salicylate;Mefenamic Acid; Menabitan Hydrochloride; Meperidine Hydrochloride;Meptazinol Hydrochloride; Methadone Hydrochloride; Methadyl Acetate;Methopholine; Methotrimeprazine; Metkephamid Acetate; MimbaneHydrochloride; Mirfentanil Hydrochloride; Molinazone; Morphine Sulfate;Moxazocine; Nabitan Hydrochloride; Nalbuphine Hydrochloride; NalmexoneHydrochloride; Namoxyrate; Nantradol Hydrochloride; Naproxen; NaproxenSodium; Naproxol; Nefopam Hydrochloride; Nexeridine Hydrochloride;Noracymethadol Hydrochloride; Ocfentanil Hydrochloride; Octazamide;Olvanil; Oxetorone Fumarate; Oxycodone; Oxycodone Hydrochloride;Oxycodone Terephthalate; Oxymorphone Hydrochloride; Pemedolac;Pentamorphone; Pentazocine; Pentazocine Hydrochloride; PentazocineLactate; Phenazopyridine Hydrochloride; Phenyramidol Hydrochloride;Picenadol Hydrochloride; Pinadoline; Pirfenidone; Piroxicam Olamine;Pravadoline Maleate; Prodilidine Hydrochloride; Profadol Hydrochloride;Propirarn Fumarate; Propoxyphene Hydrochloride; Propoxyphene Napsylate;Proxazole; Proxazole Citrate; Proxorphan Tartrate; PyrrolipheneHydrochloride; Remifentanil Hydrochloride; Salcolex; SalethamideMaleate; Salicylamide; Salicylate Meglumine; Salsalate; SodiumSalicylate; Spiradoline Mesylate; Sufentanil; Sufentanil Citrate;Talmetacin; Talniflumate; Talosalate; Tazadolene Succinate; Tebufelone;Tetrydamine; Tifurac Sodium; Tilidine Hydrochloride; Tiopinac;Tonazocine Mesylate; Tramadol Hydrochloride; Trefentanil Hydrochloride;Trolamine; Veradoline Hydrochloride; Verilopam Hydrochloride;Volazocine; Xorphanol Mesylate; Xylazine Hydrochloride; ZenazocineMesylate; Zomepirac Sodium; Zucapsaicin., Aflyzosin Hydrochloride;Alipamide; Althiazide; Amiquinsin Hydrochloride; Amlodipine Besylate;Amlodipine Maleate; Anaritide Acetate; Atiprosin Maleate; Belfosdil;Bemitradine; Bendacalol Mesylate; Bendroflumethiazide; Benzthiazide;Betaxolol Hydrochloride; Bethanidine Sulfate; Bevantolol Hydrochloride;Biclodil Hydrochloride; Bisoprolol; Bisoprolol Fumarate; BucindololHydrochloride; Bupicomide; Buthiazide: Candoxatril; Candoxatrilat;Captopril; Carvedilol; Ceronapril; Chlorothiazide Sodium; Cicletanine;Cilazapril; Clonidine; Clonidine Hydrochloride; Clopamide;Cyclopenthiazide; Cyclothiazide; Darodipine; Debrisoquin Sulfate;Delapril Hydrochloride; Diapamide; Diazoxide; Dilevalol Hydrochloride;Diltiazem Malate; Ditekiren; Doxazosin Mesylate; Eeadotril; EnalaprilMaleate; Enalaprilat; Enalkiren; Endralazine Mesylate; Epithiazide;Eprosartan; Eprosartan Mesylate; Fenoldopam Mesylate; FlavodilolMaleate; Flordipine; Flosequinan; Fosinopril Sodium; Fosinoprilat;Guanabenz; Guanabenz Acetate; Guanacline Sulfate; Guanadrel Sulfate;Guancydine; Guanethidine Monosulfate; Guanethidine Sulfate; GuanfacineHydrochloride; Guanisoquin Sulfate; Guanoclor Sulfate; GuanoctineHydrochloride; Guanoxabenz; Guanoxan Sulfate; Guanoxyfen Sulfate;Hydralazine Hydrochloride; Hydralazine Polistirex; Hydroflumethiazide;Indacrinone; Indapamide; Indolaprif Hydrochloride; Indoramin; IndoraminHydrochloride; Indorenate Hydrochloride; Lacidipine; Leniquinsin;Levcromakalim; Lisinopril; Lofexidine Hydrochloride; Losartan Potassium;Losulazine Hydrochloride; Mebutamate; Mecamylamine Hydrochloride;Medroxalol; Medroxalol Hydrochloride; Methalthiazide; Methyclothiazide;Methyldopa; Methyldopate Hydrochloride; Metipranolol; Metolazone;Metoprolol Fumarate; Metoprolol Succinate; Metyrosine; Minoxidil;Monatepil Maleate; Muzolimine; Nebivolol; Nitrendipine; Ofornine;Pargyline Hydrochloride; Pazoxide; Pelanserin Hydrochloride; PerindoprilErbumine; Phenoxybenzamine Hydrochloride; Pinacidil; Pivopril;Polythiazide; Prazosin Hydrochloride; Primidolol; PrizidilolHydrochloride; Quinapril Hydrochloride; Quinaprilat; QuinazosinHydrochloride; Quinelorane Hydrochloride; Quinpirole Hydrochloride;Quinuclium Bromide; Ramipril; Rauwolfia Serpentina; Reserpine;Saprisartan Potassium; Saralasin Acetate; Sodium Nitroprusside;Sulfinalol Hydrochloride; Tasosartan; Teludipine Hydrochloride;Temocapril Hydrochloride; Terazosin Hydrochloride; Terlakiren;Tiamenidine; Tiamenidine Hydrochloride; Tierynafen; Tinabinol;Tiodazosin; Tipentosin Hydrochloride; Trichlormethiazide; TrimazosinHydrochloride; Trimethaphan Camsylate; Trimoxamine Hydrochloride;Tripamide; Xipamide; Zankiren Hydrochloride; Zofenoprilat Arginine.,Alclofenac; Alclometasone Dipropionate; Algestone Acetonide; AlphaAmylase; Ameinafal; Ameinafide; Amfenac Sodium; AmipriloseHydrochloride; Anakinra; Anirolac; Anitrazafen; Apazone; BalsalazideDisodium; Bendazac; Benoxaprofen; Benzydamine Hydrochloride; Bromelains;Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone; Cliprofen;Clobetasol Propionate; Clobetasone Butyrate; Clopirac; CloticasonePropionate; Cormethasone Acetate; Cortodoxone; Deflazacort; Desonide;Desoximetasone; Dexamethasone Dipropionate; Diclofenac Potassium;Diclofenac Sodium; Diflorasone Diacetate; Diflumidone Sodium;Diflunisal; Difluprednate; Diftalone; Dimethyl Sulfoxide; Drocinonide;Endrysone; Enlimomab; Enolicam Sodium; Epirizole; Etodolac; Etofenamate;Felbinac; Fenamole; Fenbufen; Fenclofenac; Fenclorac; Fendosal;Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenamic Acid;Flumizole; Flunisolide Acetate; Flunixin; Flunixin Meglumine; FluocortinButyl; Fluorometholone Acetate; Fluquazone; Flurbiprofen; Fluretofen;Fluticasone Propionate; Furaprofen; Furobufen; Halcinonide; HalobetasolPropionate; Halopredone Acetate; Ibufenac; Ibuprofen; IbuprofenAluminum; Ibuprofen Piconol; Ilonidap; Indomethacin; IndomethacinSodium; Indoprofen; Indoxole; Intrazole; Isoflupredone Acetate;Isoxepac; Isoxicam; Ketoprofen; Lofemizole Hydrochloride; Lornoxicam;Loteprednol Etabonate; Meclofenamate Sodium; Meclofenamic Acid;Meclorisone Dibutyrate; Mefenamic Acid; Mesalamine; Meseclazone;Methylprednisolone Suleptanate; Momiflumate; Nabumetone; Naproxen;Naproxen Sodium; Naproxol; Nimazone; Olsalazine Sodium; Orgotein;Orpanoxin; Oxaprozin; Oxyphenbutazone; Paranyline Hydrochloride;Pentosan Polysulfate Sodium; Phenbutazone Sodium Glycerate; Pirfenidone;Piroxicam; Piroxicam Cinnamate; Piroxicam Olamine; Pirprofen;Prednazate; Prifelone; Prodolic Acid; Proquazone; Proxazole; ProxazoleCitrate; Rimexolone; Romazarit; Salcolex; Salnacedin; Salsalate;Sanguinarium Chloride; Seclazone; Sermetacin; Sudoxicam; Sulindac;Suprofen; Talmetacin; Talniflumate; Talosalate; Tebufelone; Tenidap;Tenidap Sodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine; Tiopinac;Tixocortol Pivalate; Tolmetin; Tolmetin Sodium; Triclonide;Triflumidate; Zidometacin; and Zomepirac Sodium.

In an embodiment, the CMBPC may contain additional ingredients assuitable for the formulation of a pharmaceutical composition. As usedherein, “additional ingredients” include, but are not limited to, one ormore of the following: excipients; surface active agents; dispersingagents; inert diluents; granulating and disintegrating agents; bindingagents; lubricating agents; sweetening agents; flavoring agents;coloring agents; preservatives; physiologically degradable compositionssuch as gelatin; aqueous vehicles and solvents; oily vehicles andsolvents; suspending agents; dispersing or wetting agents; emulsifyingagents, demulcents; buffers; salts; thickening agents; fillers;emulsifying agents; antioxidants; antibiotics; antifungal agents;stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials.

Generally, the CMBPC may be administered systemically in dosage unitformulations containing conventional nontoxic pharmaceuticallyacceptable carriers, adjuvants, and vehicles as desired, or may belocally administered by means such as, but not limited to, injection,implantation, or grafting. In a specific embodiment, administration ofthe CMBPC may be by intravenous injection, endobronchial adminstration,intraaterial injection, intramuscular injection, intracardiac injection,subcutaneous injection, intraperitoneal injection, intraperitonealinfusion, transdermal diffusion, transmucosal diffusion, intracranial,intrathecal, or combinations thereof. A means of administering the CMBPCmay include, but is not limited to, infusion. Systemically may alsoinclude, for example, by a pump, by an intravenous line, or by bolusinjection. Bolus injection can include subcutaneous, intramuscular, orintraperitoneal routes.

Although the descriptions of pharmaceutical (e.g., CMBPC) andprophylactic compositions provided herein are principally directed topharmaceutical compositions which are suitable for ethicaladministration to humans, it will be understood by the skilled artisanthat such compositions are generally suitable for administration toanimals of all sorts. Modification of compositions suitable foradministration to humans of the type disclosed herein in order to renderthe compositions suitable for administration to various animals can beaccomplished by the ordinarily skilled veterinary pharmacologist, withthe benefit of this disclosure, who can design and perform suchmodifications with routine, if any, experimentation. Subjects to whichadministration of the pharmaceutical compositions of this disclosure iscontemplated include, but are not limited to, humans and other primates;mammals including commercially relevant mammals such as cattle, pigs,horses, and sheep; companion animals such as cats, and dogs; and birdsincluding commercially relevant birds such as chickens, ducks, geese,and turkeys.

In certain embodiments, a therapeutically effective dose of the CMBPCand/or RC is delivered to the subject. A therapeutically effective dosewill be determined using a variety of factors (e.g., the body weight ofthe subject) and may be further modified, for example, based on theseverity or phase of the medical condition. The number of cells in theCMBPC and/or RC used will depend on the weight and condition of thereceiver subject, the number of or frequency of administrations, andother variables. For example, a therapeutic dose may be one or moreadministrations of the CMBPC and/or RC.

In an embodiment, the CMBPC and/or RC are formulated for topicaladministration into forms such as creams, lotions, serums, powders,ointments, or drops. A formulation of CMBPC and/or RC for topicaladministration may also contain pharmaceutically acceptable carriers,moisturizers, oils, fats, waxes, surfactants, thickening agents,antioxidants, viscosity stabilizers, chelating agents, buffers,preservatives, perfumes, dyestuffs, lower alkanols, humectants,emollients, dispersants, sunscreens such as radiation blocking compoundsor UV-blockers, antibacterials, antifungals, disinfectants, vitamins,antibiotics, anti-acne agents, as well as other suitable materials thatdo not have a significant adverse effect on the activity of the topicalcomposition. Nonlimiting exemplary pharmaceutically acceptable carriersthat may be used in the compositions comprising the restored cells mayinclude water, mixtures of water and water-miscible solvents such aslower alkanols or vegetable oils, and water-soluble ophthalmologicallyacceptable non-toxic polymers (for example, cellulose derivatives suchas methylcellulose), glycerin, propylene glycol, methylparaben,alginates, glyceryl stearate, PEG-100 stearate, cetyl alcohol,propylparaben, butylparaben, sorbitols, polyethoxylated anhydrosorbitolmonostearate (TWEEN®), white petrolatum (VASELINE®), triethanolamine,emu oil, aloe vera extract, lanolin, cocoa butter, LIPODERM® base, andthe like. In an embodiment, the CMBPC and/or RC formulated for topicaladministration may be applied to one or more areas of the skin includingthe face, hands, and neck.

In an embodiment, the methodologies disclosed herein result in therapiesthat are prophylactic, palliative, curative, or combinations thereof.Methodologies and compositions of the type disclosed herein may beutilized in the treatment of a wide variety of medical conditionsrelated to decreases in cellular function and viability such asage-related medical conditions that include neurological disorders;autoimmune diseases; and disorders associated with radiationoverexposure (chronic or acute).

It is contemplated the methodologies and compositions disclosed hereinmay result in an increased expression of genes associated with improvedcellular health with a concomitant decrease in the expression of genesassociated with adverse cellular events. In some embodiments, themethodologies and compositions disclosed herein result in an increasedexpression of genes associated with beneficial cellular events.

According to another aspect of the disclosure, kits are provided. Kits,according to the present disclosure, include package(s) or containerscomprising the compositions disclosed herein (e.g., RC, cell-freeculture media) and may include defined culture medium and cell culturemedium supplement. The kit may further include an instruction letter orpackage-associated instruction for the treatment and/or prophylaxis of amedical condition. The phrase “package” means any vessel containing thecompositions (including stem cells, media, and/or media supplement)presented herein. For example, the package can be a box or wrapping.Examples of pharmaceutical packaging materials include, but are notlimited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials,containers, syringes, bottles, and any packaging material suitable for aselected formulation and intended mode of administration and treatment.The kit can also contain items that are not contained within the packagebut are attached to the outside of the package, for example, pipettes.Kits may optionally contain instructions for administering compositionsof the present disclosure to a subject having a condition in need oftreatment. Kits may also comprise instructions for approved uses ofcompounds herein by regulatory agencies, such as the United States Foodand Drug Administration. Kits may optionally contain labeling or productinserts for the present compositions. The package(s) and/or any productinsert(s) may themselves be approved by regulatory agencies. The kitscan include compounds in the solid phase or in a liquid phase (such asbuffers provided) in a package. The kits also can include buffers forpreparing solutions for conducting the methods, and pipettes fortransferring liquids from one container to another. The kit mayoptionally also contain one or more other compounds for use incombination therapies as described herein. In certain embodiments, thepackage(s) is a container for intravenous administration.

In an embodiment, a subject having undergone a restoration method of thetype disclosed herein may be subsequently monitored for some timeperiod. Monitoring of the subject may comprise qualitative andquantitative evaluations of the subject's general health and/or medicalcondition. In some embodiments, a subject may be subjected to aplurality of cellular restoration methods of the type disclosed herein.For example, a receiver subject having undergone a cellular restorationmethod of the type disclosed herein may display quantitative and/orqualitative improvements in the subject's general health and/or medicalcondition for some time period. Subsequently, the receiver subject mayexperience some decline in their general health and/or medical conditionand another cellular restoration process may be carried out. Thecellular restoration process may involve obtaining a donor cell sampleand/or receiver cell sample utilizing the methodologies disclosedherein, performing cellular restoration of the receiver cell sample andadministering the restored cell sample to the receiver subject.Alternatively, the receiver subject may be administered at least aportion of the restored cell sample remaining from a prior cellularrestoration process.

In some embodiments, evaluations of the subject comprise determinationsbased on analyses disclosed herein (e.g., natural killer assay, telomerelength, gene and protein biomarker arrays). In such embodiments, thesubject may provide a receiver cell sample and the quality of the sampleevaluated as disclosed herein. In some embodiments, the receiver cellsample quality value at some point post-restoration may be compared tothe receiver cell sample quality value pre-restoration and thisinformation utilized to assess whether additional treatment is needed.For example, a subject having a receiver cell sample pre-restorationquality value of 5 may have a receiver cell sample post-restorationquality value of 9 for a time period of up to about 1 year subsequent tothe restoration process. The subject's post-restoration receiver cellsample quality value after 1.5 years may have decreased to 7 while after3 years the value may be 5. In such instances, the subject may beadministered another RC.

In an embodiment, a method for cellular restoration comprises theisolation of one or more constituents present in the culture media of adonor cell sample. Such materials may be investigated for their abilityto restore cellular functions of the type disclosed herein to a receivercell sample. In an embodiment, the IREs comprise vesicular bodies ormaterials derived from vesicular bodies such as exosomes, exosomecomplexes, proteins, cytokines, microvesicles, microvesicle complexes,microRNAs, large non-coding RNAs or combinations thereof. Exosomes arecomposed of an evolutionary-conserved common set of protein moleculesthat vary with the cell and tissue of origin, as well as mRNA andnon-coding RNA (ncRNA) molecules, such as microRNA (miRNA) and longnon-coding RNA (lncRNA). Exosomes function as mediators of extracellularsignaling, via the membrane-protected transfer of cellular material andmay play a variety of roles such as induction of tolerance againstallergen, eradication of established tumors in mice, and the inhibitionand activation of natural killer cells. As will be understood by one ofordinary skill in the art, exosomes and microvesicles can be envisionedas carriers of biomolecule cargo (e.g, RNA, proteins). Hereinafter, forsimplicity, carriers isolated from a donor cell sample with the abilityto restore cellular functions of the type disclosed herein to a receivercell sample (e.g., exosomes) are termed collectively termed isolatedrestoring elements (IRE) while the cargo present in such IREs (e.g.,RNA) are collectively termed molecules of cellular restoration (MCR).

IREs may be identified from the culture media (CM) of a donor cellsample using any suitable methodology. For example, IREs may be isolatedfrom CM through either ultracentrifugation or precipitation/low speedcentrifugation with commercially available products such as exosomeisolation kits.

In an embodiment, the IRE is a microvesicle also known as microparticlesor ectosomes. Microvesicles are fragments of plasma membranes that areusually larger than 0.2 μm in size which are shed from all cell types.Microvesicles also play a role in intracellular communication and cantransport mRNA, miRNA, proteins, and lipids between cells. In anembodiment, an RC of the type disclosed herein is a mixture comprisingisolated exosomes and a pharmaceutically acceptable carrier formulatedfor administration to a subject. In an embodiment, an RC of the typedisclosed herein is a mixture comprising isolated microvesicles and apharmaceutically acceptable carrier formulated for administration to asubject.

In an embodiment, isolated exosomes and/or isolated microvesicles may beutilized in the compositions and methodologies disclosed herein.Alternatively, isolated exosomes and/or isolated microvesicles may befurther subjected to methodologies to isolate and/or characterizematerials associated with the exosomes and/or microvesicles.

In an embodiment, the methods disclosed herein further comprise analysisof the cargo (i.e., biochemical composition) of the isolated exosomesand/or microvesicle (i.e., MCR). Any suitable methodology may beemployed for determining the biochemical composition of the exosomesand/or microvesicles. For example, exosomal or microvesical material maybe extracted and the extracted material subjected to characterization bytechniques such as sequencing and gel analysis. Isolated exosomal and/ormicrovesical material may be investigated to determine the ability ofthese materials to effect cellular restoration in a manner similar tothose disclosed previously herein for the RCs. In an embodiment one ormore constituents of the biochemical composition of an isolated exosomeand/or microvesicle are determined to effect the cellular restoration ofa receiver cell sample.

In an embodiment, an MCR of the type disclosed herein may compriseisolated macromolecules such as proteins, genomic DNA (gDNA),mitochondrial DNA (mtDNA, major histocompatibility complex (MHC),polysaccharides, cytokines, lipids, nucleic acids, RNA, or combinationsthereof. Alternatively, an MCR of the type disclosed herein may comprisea small molecule such as a metabolite, or natural product. In anembodiment, the MCR may be sourced from cellular material (e.g., a donorcell sample). Alternatively, the MCR may be recombinant in nature in thecase of macromolecules or may be synthetic in the case of smallmolecules. An embodiment of a composition suitable for use in thepresent disclosure comprises an MCR formulated for administration to asubject in need thereof. Such materials may be administered in a mannersimilar to those disclosed for administration of one or more embodimentsof RCs. For example, a formulation suitable for administration to asubject in need thereof may comprise an IRE and/or MCR and one or moreof the pharmaceutically active materials disclosed herein (e.g.,antibiotics).

IREs and/or MCRs for parenteral injection may comprise pharmaceuticallyacceptable sterile aqueous or nonaqueous solutions, dispersions,suspensions or emulsions and sterile powders for reconstitution intosterile injectable solutions or dispersions. Examples of suitableaqueous and nonaqueous carriers, diluents, solvents or vehicles includewater, ethanol, polyols (propylene glycol, polyethylene glycol,glycerol, and the like), suitable mixtures thereof, vegetable oils (suchas olive oil) and injectable organic esters such as ethyl oleate. Properfluidity may be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants.

Compositions comprising the IREs and/or MCRs may also containpharmaceutically acceptable carriers, moisturizers, oils, fats, waxes,surfactants, thickening agents, antioxidants, viscosity stabilizers,chelating agents, buffers, preservatives, perfumes, dyestuffs, loweralkanols, humectants, emollients, dispersants, sunscreens such asradiation blocking compounds or UV-blockers, antibacterials,antifungals, disinfectants, vitamins, antibiotics, anti-acne agents, aswell as other suitable materials that do not have a significant adverseeffect on the activity of the topical composition. Nonlimiting exemplarypharmaceutically acceptable carriers that may be used in thecompositions comprising the IRE may include water, mixtures of water andwater-miscible solvents such as lower alkanols or vegetable oils, andwater-soluble ophthalmologically acceptable non-toxic polymers (forexample, cellulose derivatives such as methylcellulose), glycerin,propylene glycol, methylparaben, alginates, glyceryl stearate, PEG-100stearate, cetyl alcohol, propylparaben, butylparaben, sorbitols,polyethoxylated anhydrosorbitol monostearate (TWEEN), white petrolatum(VASELINE), triethanolamine, emu oil, aloe vera extract, lanolin, cocoabutter, LIPODERM base, and the like.

Prevention of the action of microorganisms may be effected by theinclusion by various antibacterial and antifungal agents in any of thecompositions disclosed herein for example, parabens, chlorobutanol,phenol, sorbic acid, and the like. It may also be desirable to includeisotonic agents, for example, sugars, sodium chloride and the like.

In an embodiment, the IREs and/or MCRs are formulated as compositionsfor topical administration. Such topical restoring compositions mayinclude additives suitable for the formulation of a composition fortopical application. In an embodiment, the topical restoring compositionmay be applied to one or more areas of the skin including the face,hands and neck.

EXAMPLES

The subject matter of the present disclosure having been generallydescribed, the following examples are given as particular embodiments ofthe disclosure and to demonstrate the practice and advantages thereof.It is understood that the examples are given by way of illustration andare not intended to limit the specification or the claims to follow inany manner.

Example 1

Sample and Techniques

Peripheral blood mononuclear cells were collected from subjects havingundergone a Stage B preparation involving the administration of themobilizer NEUPOGEN® (filagrastim). For the following experimentsperipheral blood mononuclear cells (1×5 mL vial per subject) were foundto contain the following number of nucleated cells:

Approximate Subject Age Sex Cell Number R1 70 Male 1.8 × 10⁸ R2 70 Male2.7 × 10⁸ R3 60 Male 2.2 × 10⁸ D1 26 Male 3.3 × 10⁸ D2 30 Male 1.8 × 10⁸D3 28 Female 1.9 × 10⁸

The cells following harvest were cryopreserved and upon thawing weredetermined by trypan blue dye exclusion and flow cytometry to be greaterthan 95%.

The percentages of stem cell, progenitor cell and mature cellpopulations were determined by flow cytometry. Table 3 shows the averagepercentage of hematopoietic stem cells (HSCs) and early hematopoieticprogenitor cells (HPCs) to be ˜0.5% and 1.0% respectively of the totalmobilized cell collection.

TABLE 3 Hematopoietic Hematopoietic Progenitor Stem Cells CellsHematopoietic CD34⁺ CD34⁺ T Cells NK Cells Cells CD45⁺ CD38⁻ CD38⁺ CD3⁺CD56⁺ % Total Cell Population R1 64.4 0.5 0.3 39.6 4.2 R2 59.9 0.4 1.629.7 7.5 R3 86.3 0.4 0.3 56.7 2.9 D1 64.0 1.0 1.5 32.4 5.7 D2 59.9 0.30.8 36.0 4.1 D3 59.0 4.1 1.0 10.5 5.5 % Hematopoietic (CD45⁺) PopulationR1 100 0.7 0.4 61.5 6.5 R2 100 0.7 2.8 49.5 12.5 R3 100 0.4 0.4 65.7 3.4D1 100 1.5 2.4 50.5 8.9 D2 100 0.5 1.3 60.1 6.9 D3 100 7.0 1.7 51.7 9.3

TABLE 4 Mesenchymal Endothelial Stem Cells Progenitor Non- CD29⁺ CellsHematopoietic CD44⁺ CD31⁺ Cells CD45⁻ CD105⁺ CD105⁺ % Total CellPopulation R1 17.9 0.11 0.06 R2 38.7 0.91 0.79 R3 11.1 0.22 0.18 D1 33.40.52 0.44 D2 26.7 0.69 0.65 D3 40.4 0.63 0.46 % Non- Hematopoietic(CD45⁻) Population R1 100 0.60 0.35 R2 100 2.34 2.05 R3 100 1.94 1.58 D1100 1.57 1.32 D2 100 2.57 2.43 D3 100 1.56 1.14

Receiver cell samples (i.e., R1, R2, and R3) were individually pairedwith donor cell samples (i.e., D1, D2, and D3) and the pairs co-culturedin a transwell culture cell for four weeks. The morphology of the cellswas studied at 2 weeks into the co-culture and at 4 weeks of co-culture.Gene expression arrays, protein arrays, and telomere length experimentscompared freshly defrosted cells from the collection tubes (referred toas the baseline donor cell sample or baseline receiver cell sample) withcells at the 4-week study endpoint (referred to as restored cellsamples).

At the midpoint of the co-culture (i.e., 2 weeks) baseline receiver cellsamples displayed morphologies consistent with low viability. Restoredcell samples displayed a robust cellular morphology that included colonyformation.

Example 2

Protein array analyses were carried out using conditioned media from thebaseline donor cell sample or baseline receiver cell sample. Theconditioned media was mixed with like cellular protein extracts andapplied to the custom-designed arrays which consisted of antibody probesfor 68 factors linked to cellular aging and senescence, collectivelyreferred to as the senescence-associated secretory factors (SASF).Quantitative PCR gene array analyses were carried out by extracting RNAfrom the baseline samples or the co-cultured donor and receiver samples.The data presented represents the average metric determined for eitherthe baseline donor cell sample, the baseline receiver cell sample, orthe restored cell sample.

The results of the gene array analyses demonstrated that there was lessthan a 2-fold difference in the majority of senescence-related genes forthe baseline donor cell samples and the baseline receiver cell samples,FIGS. 3 and 4. In all of the figures presented as plots of the geneexpression analysis, the squares represent genes which are expressed ata lower level following the cellular restoration, triangles representgenes expressed at a higher level following restoration and circlesrepresent genes whose expression level was determined to besubstantially similar to the expression level prior to cellularrestoration. The designation of “substantially similar” is qualitativeand reflects the close proximity of the value to the line. The datasuggests the techniques disclosed herein for the mobilization andcollection of donor cell samples and receiver cell samples select fornon-senescent cells. The results of the protein-based arrays, whichassessed levels of senescence-related factors produced either within thecells or released into the culture media, similarly displayed littledifference between the senescence-related factors among the donor cellsample and receiver cell sample, FIG. 5. The mean telomere lengthbetween the donor cell samples and receiver cell samples were not foundto be significantly different, FIG. 6.

The results demonstrate that the gene expression profiles and proteinexpression profiles of the donor cell samples and receiver cell sampleswere similar despite the difference in age of the subjects. Further,despite the difference in age between the donor subjects and receiversubjects, the cells had similar telomere lengths.

Example 3

The restored cell samples were investigated using the gene and proteinarrays of Example 2 and are shown in FIGS. 7 and 8, respectively. Foreach of the restored cell samples investigated, approximately half ofthe examined genes (as designated by the “Xs” on the Figure) wereexpressed at a lower level in the restored cell samples when compared tothe baseline donor cell sample. The genes that were expressed at a lowerlevel were genes associated with improving cellular function anddecreasing the extent of cellular senescence and aging. The datasuggests the gene expression profile of the restored cell samples werealtered by the transwell restoration and more closely approximated thatof the baseline donor cell sample than that of the baseline receivercell sample. The data demonstrate the restore cell sample exhibited adecreased expression of senescence-related genes of receiver cellsand/or cell types compared to the receiver cell sample, whereinsenescence-related genes are defined as the RBGEP, by quantitativepolymerase chain reaction as shown in FIGS. 7 and 8.

Further, examination of the clustering analyses revealed a subset ofgenes (designated gene set A) whose expression was consistently elevatedin the baseline receiver cell sample but whose expression was reduced inthe restored cell sample. The expression of Gene Set A in the restoredcell sample was reduced to levels comparable to those observed in thebaseline donor cell sample. Similarly, as shown in FIG. 9A,stratification of the protein arrays identified 13 factors that showed asimilar elevated trend in the baseline receiver cell samples whencompared to that of the baseline donor cell samples. Likewise, as shownin FIG. 9B, the restored cell samples exhibit a level of expression ofthe identified 13 factors comparable to that observed in the baselinedonor samples. These findings demonstrate a methodology for monitoringthe restoration of the receiver cell sample by gene and protein arrayanalyses. Further, these data demonstrate the restored cell sampleexhibited a decreased expression of senescence-associated secretoryfactors compared to the receiver cell sample at baseline, whereinsenescence-associated secretory factors are defined in Table 1, asmeasured by antibody array, as in FIGS. 9, 10 and 11, or enzyme-linkedimmunosorbant assay.

Example 4

Gene and protein arrays of restored cell samples for individual pairs ofbaseline donor cell samples and receiver cell samples were investigated.Specifically receiver cell sample R1 was co-cultured in a transwellexperiment with donor cell samples from D1 (FIG. 10B), D2 (FIG. 10C),and D3 (FIG. 10D) respectively. Hierarchical clustergrams showed thatall of the genes investigated are elevated in the receiver cell sample(FIG. 10A) while these same genes are expressed at low to modest levelsin D1, D2 and D3. The restored cell sample was found to have a geneexpression comparable to that of the level of expression observed forD1, D2, and D3. Experiments carried out using receiver cell samples R2or R3 with donor cell samples D1, D2, or D3 exhibited similar results.

Example 5

The nature of the soluble particles passing through the permeablemembrane in the transwell co-culture experiments was investigated.Specifically, transwell co-culture experiments were carried out in thepresence or absence of manumycin. Manumycin,-[(1S,5S,6R)-5-hydroxy-5-[(1E,3E,5E)-7-[(2-hydroxy-5-oxo-1-cyclopenten-1-yl)amino]-7-oxo-1,3,5-heptatrien-1-yl]-2-oxo-7-oxabicyclo[4.1.0]hept-3-en-3-yl]-2E,4E,6R-trimethyl,2,4-decadienamide, is an antibiotic that acts a potent and selectivefarnesytransferase inhibitor. Mannumycin is also known to inhibit therelease of exosomes. Restored cells co-cultured in the presence of 5 μMmanumycin displayed less robust morphology than restored cells culturedin the absence of manumycin. Gene expression analysis of restored cellsamples co-cultured in the presence of manumycin did not display achange in expression levels similar to those observed in the absence ofmanumycin. In contrast, protein expression analyses of restored cellsamples co-cultured in the presence of manumycin found elevated levelsof all proteins investigated when compared to the proteins levels forrestored cell samples co-cultured in the absence of manumycin, FIG. 11.The data suggests the role of exosome/microvesicles in mediating thedisclosed cellular restoration process. Further support for the role ofexosome/microvesicles in mediating the disclosed cellular restorationprocess is shown in FIG. 12. FIG. 12 displays the telomere length for arestored cell sample co-cultured in the presence or absence ofmanumycin. The telomere length in the presence of manumycin is decreasedsuggesting exosome/microvesicles play a role in the restoration process.

Example 6

The innate immune function of the baseline donor cell samples andreceiver cell samples were evaluated using a natural killer cell assay,FIG. 13. The assay was also performed on restored samples, FIG. 14. Therestored cell samples are identified by the receiver cell sample-donorcell sample that were contacted, for example, receiver cell sample 3 anddonor cell sample D2 are listed as R3-D2. The data demonstrate that therestored cell samples R1-D3, R1-D2, R3-D2, and R2-D2 maintained properimmune function while restored cell sample R1-D1 had decreased immunefunction. The data illustrate that for D2 and D3 the restored cellsamples are characterized by an improvement in cellular immune functionas quantified by natural killer cell cytotoxicity assay, as illustratedin FIGS. 13 and 14, and/or T-cell mitogen response assay.

The hematopoietic function of the baseline donor cell samples andreceiver cell samples were evaluated using a clonogenic assay, FIG. 15.The data demonstrate that the restored cell samples R1-D3, R1-D2, R3-D2,and R2-D2 maintained proper hematopoietic function while restored cellsample Rl-D1 had decreased hematopoietic function. The data illustratethat the restored cell sample is characterized by an improvement incellular hematopoietic function as quantified by hematopoietic stem cellclonogenic assay, as observed in FIG. 15.

Cell population analyses were performed by flow cytometry to investigatewhether the original distribution of cell types in the receiver cellsample, R1, was altered by the 4-week restoration process with donorcell sample D2. The results, shown in FIGS. 16A-16D, demonstrated thatalthough there was some loss in the percentage of endothelial progenitorcells (FIG. 16A), there was an expansion of the mesenchymal stem cellcompartment (FIG. 16B). As shown in FIG. 16C, a combination of thepercentages of non-hematopoietic cells (i.e., from FIGS. 16A and B)indicated that the total percentages of these populations weremaintained during the restoration process. Similarly, as shown in FIG.16D, the percentages of hematopoietic stem and progenitor cells wereinsignificantly changed. These data demonstrate that the restoredcomposition prepared by the methodologies disclosed herein ischaracterized by a lack of change in the percentage of hematopoieticstem cells, hematopoietic progenitor cells, mesenchymal stem cells andendothelial progenitor cells, herein termed the “stem cell pool”, inreceiver cells after cellular restoration compared to receiver samplesat baseline, as observed in FIG. 16, by flow cytometry.

Example 7

The role of exosomes in cellular restoration was investigated. Exosomeswere collected from cells that were cultured in exosomes-free media. Thecomplete media containing sera were cleared of exosomes by overnightultracentrifugation at 100,000 g and then stored at 40 C. Media werecollected and then subjected to differential ultracentrifugation toisolate exosomes. The media was cleared of cells by centrifugation at2,000 g for 20 min. The supernatant was transferred to a sterile tubefor centrifugation at 10,000 g for 30 min to remove remaining celldebris and large vesicles. The supernatant was transferred toultracentrifuge tubes and spun at 100,000 g for 80 min. The supernatantwas disposed and the exosome pellet was washed with PBS. A final spinwas done at 100,000 g for 80 min and the exosome pellet was eitherresuspended in 100 μl PBS for TEM or for the extraction of RNA. Exosomecollection from smaller volumes was performed with the Total ExosomeIsolation kit from Invitrogen. The isolation protocol followedmanufacturer's guidelines. Collected exosomes washed once with PBS.Exosome samples were diluted in PBS to a final protein concentration of100 μg/ml. In brief, 20 μl of exosome solution was placed on formavarcarbon coated grids. The solution was wicked off the grid with filterpaper and 20 μl of PTA (phosptungstaic acid) stain solution was thenplaced on the grid and wicked off with filter paper. The grid was scopedon a JEOL 1200EX electron microscope and digital images were obtainedusing an Advanced Microscopy Techniques (AMT camera) (Woburn, Mass.).

The following enumerated embodiments are provided as non-limitingexamples.

A first embodiment which is a method comprising (i) obtaining a firstcell sample from a first subject; (ii) obtaining a second cell samplefrom a second subject; (iii) culturing the first cell sample in thepresence of at least a portion of a culture media of the second cellsample for a time period ranging from about 24 hours to about 6 weeks toproduce a restoring composition; and (iv) contacting the restoringcomposition with the second cell sample for a period of time rangingfrom about 24 hours to about 6 weeks to produce a restored composition.

A second embodiment which is the method of the first embodiment furthercomprising immunophenotyping the first cell sample, the second cellsample, the restored composition, or combinations thereof.

A third embodiment which is the method of any of the first throughsecond embodiments wherein the first subject, the second subject, orboth are related by consanguinity.

A fourth embodiment which is the method of any of the first throughthird embodiments wherein the first subject and the second subjectdiffer in chronological age by from about 5 years to about 75 years.

A fifth embodiment which is the method of any of the first throughfourth embodiments wherein the second subject has a medical conditionthat is undiagnosed in the first subject.

A sixth embodiment which is the method of any of the first through fifthembodiments wherein the first subject, the second subject, or both havebeen administered a mobilizer prior to step (i).

A seventh embodiment which is the method of any of the first throughsixth embodiments wherein the first cell sample and second cell samplecomprise adult stem cells.

An eighth embodiment which is the method of any of the first throughseventh embodiments wherein the first cell sample and second cell sampleexclude stem cells that are embryonic in origin.

A ninth embodiment which is the method of any of the first througheighth embodiments wherein the first cell sample, the second cellsample, or both are obtained from a subject's peripheral blood.

A tenth embodiment which is the method of any of the first through ninthembodiments wherein the first cell sample, the second cell sample, orboth are harvested directly from a subject's bone marrow.

An eleventh embodiment which is the method of any of the first throughtenth embodiments wherein the first cell sample, the second cell sample,or both comprise non-hematopoietic cells, mesenchymal stem cells,endothelial progenitor cells, hematopoietic stem cells, primitivehematopoietic stem cells, hematopoietic progenitor cells, differentiatedhematopoietic cells, T-lymphocytes, natural killer cells, orcombinations thereof.

A twelfth embodiment which is the method of any of the first throughtwelfth embodiments wherein the first cell sample, the second cellsample, or both comprise non-senescent and senescent cells.

A thirteenth embodiment which is the method of the twelfth embodimentwherein the non-senescent cells are present in an amount of from about90% to about 99% based on the total cell number.

A fourteenth embodiment which is the restoring composition of the firstembodiment.

A fifteenth embodiment which a kit comprising the restoring compositionof the first embodiment.

A sixteenth embodiment which is a method comprising administering to asubject an effect amount of the restoring composition of the firstembodiment.

A seventeenth embodiment which is a pharmaceutical formulationcomprising the restoring composition of the first embodiment and anactive selected from the group consisting of antimicrobials, steroids,pain medications, anti-inflammatory agents, growth factors; cytokines,hormones, and combinations thereof.

An eighteenth embodiment which is an exosome/microvesicle isolated fromthe first embodiment.

A nineteenth embodiment which is a microvesicle isolated from therestoring composition of the first embodiment.

A twentieth embodiment which is a kit comprising a microvesicle isolatedfrom the restoring composition of the first embodiment.

A twenty-first embodiment which is a kit comprising an exosome isolatedfrom the restoring composition of the first embodiment.

A twenty-second embodiment which is a method comprising administering toa subject an effect amount of an exosome isolated from the restoringcomposition of the first embodiment.

A twenty-third embodiment which is a method comprising administering toa subject an effect amount of a microvesicle isolated from the restoringcomposition of the first embodiment.

A twenty-fourth embodiment which is a pharmaceutical formulationcomprising an exosome isolated from the restoring composition of thefirst embodiment and an active selected from the group consisting ofantimicrobials, steroids; pain medications, anti-inflammatory agents,growth factors, cytokines, hormones, and combinations thereof.

A twenty-fifth embodiment which is a pharmaceutical formulationcomprising a microvesicle isolated from the restoring composition of thefirst embodiment and an active selected from the group consisting ofantimicrobials, steroids; pain medications, anti-inflammatory agents,growth factors, cytokines, hormones, and combinations thereof.

A twenty-sixth embodiment which is a plurality of exosomes isolated fromthe restoring composition of the first embodiment.

A twenty-seventh embodiment which is a plurality of microvesiclesisolated from the restoring composition of claim 1.

A twenty-eighth embodiment which is a cell free media prepared byfiltration of the restoring composition of claim 1 wherein the cell freemedia comprise extracellular vesicles.

A twenty-ninth embodiment which is a method comprising contacting areceiver cell sample with a donor cell sample to produce a restoredcomposition wherein the restored composition has an innate immunefunction as determined by a natural killer cell assay that is increasedwhen compared to the innate immune function of the receiver cell sampleand wherein the addition of mannumycin A to the donor cell sample priorto contact with the receiver cell sample inhibits the increase in innateimmune function of the restored composition.

A thirtieth embodiment which is a pharmaceutical formulation comprisingexosomes derived from mobilized stem cells, and one or morepharmaceutically acceptable carriers, adjuvants, or vehicles.

A thirty-first embodiment which is the formulation of the thirtiethembodiment further comprising an agent chosen from the group consistingof antimicrobials, steroids, pain medications, anti-inflammatory agents;growth factors, cytokines, hormones, and combinations thereof.

A thirty-second embodiment which is s kit comprising the formulation ofthe thirtieth embodiment.

A thirty-third embodiment which is a method comprising administering toa subject an effect amount of the formulation of the thirtiethembodiment.

A thirty-fourth embodiment which is a pharmaceutical formulationcomprising a microvesicles derived from mobilized stem cells, and one ormore pharmaceutically acceptable carriers, adjuvants, or vehicles.

A thirty-fifth embodiment which is the formulation of the thirty-fourthembodiment further comprising an agent chosen from the group consistingof antimicrobials, steroids, pain medications, anti-inflammatory agents;growth factors, cytokines, hormones, and combinations thereof.

A thirty-sixth embodiment which is a kit comprising the formulation ofthe thirty-fourth embodiment.

A thirty-seventh embodiment which is a method comprising administeringto a subject an effect amount of the formulation of the thirty-fourthembodiment.

A thirty-eighth embodiment which is a pharmaceutical formulationcomprising an exosome-derived and/or microvesicle derived molecule andone or more pharmaceutically acceptable carriers, adjuvants, orvehicles.

A thirty-ninth embodiment which is the formulation of the thirty-eighthembodiment wherein the exosome-derived and/or microvesicle derivedmolecule comprises proteins, cytokines, microvesicles, microvesiclecomplexes, microRNAs, large non-coding RNAs or combinations thereof.

A fortieth embodiment which is a kit comprising the formulation of thethirty-eighth embodiment.

A forty-first embodiment which is a method comprising administering to asubject an effect amount of the formulation of the thirty-eighthembodiment.

A forty-second embodiment which is a pharmaceutical formulationcomprising a material selected from the group consisting of recombinantproteins, recombinant nucleic acids, synthetic metabolites, andsynthetic lipids and one or more pharmaceutically acceptable carriers,adjuvants, or vehicles wherein the material can be derived from anexosome or microvesicle.

A forty-third embodiment which is the formulation of the forty-secondembodiment further comprising an agent chosen from the group consistingof antimicrobials, steroids, pain medications, anti-inflammatory agents;growth factors, cytokines, hormones, and combinations thereof.

A forty-fourth embodiment which is a kit comprising the formulation ofthe forty-second embodiment.

A forty-fifth embodiment which is a method comprising administering to asubject an effect amount of the formulation of the forty-secondembodiment.

A forty-sixth embodiment which is a method comprising (i) obtaining afirst cell sample from a first subject; (ii) culturing the cell samplein for a time period ranging from about 24 hours to about 6 weeks toproduce a restoring composition; and (iii) removing cells from thesample to produce a cell-free restoring composition.

A forty-seventh embodiment which is the method of the forty-sixthembodiment further comprising contacting the cell-free restoringcomposition with a second cell sample obtained from a second subject.

A forty-eighth embodiment which is the method of the forty-sixthembodiment wherein the first subject, the second subject, or both arerelated by consanguinity.

A forty-ninth embodiment which is the method of the forty-sixthembodiment wherein the first subject and the second subject differ inchronological age by from about 5 years to about 75 years.

While embodiments of the present disclosure have been shown anddescribed, modifications thereof can be made by one skilled in the artwithout departing from the spirit and teachings of the disclosure. Theembodiments described herein are exemplary only, and are not intended tobe limiting. Many variations and modifications of the disclosure arepossible and are within the scope of the invention. Use of the term“optionally” with respect to any element of a claim is intended to meanthat the subject element is required, or alternatively, is not required.Both alternatives are intended to be within the scope of the claim. Useof broader terms such as comprises, includes, having, etc. should beunderstood to provide support for narrower terms such as consisting of,consisting essentially of, comprised substantially of, etc.

Accordingly, the scope of protection is not limited by the descriptionset out above but is only limited by the claims which follow, that scopeincluding all equivalents of the subject matter of the claims. Each andevery claim is incorporated into the specification as an embodiment ofthe present invention. Thus, the claims are a further description andare an addition to the preferred embodiments of the present invention.The discussion of a reference in the Background is not an admission thatit is prior art to the present invention, especially any reference thatmay have a publication date after the priority date of this application.The disclosures of all patents, patent applications, and publicationscited herein are hereby incorporated by reference, to the extent thatthey provide exemplary, procedural or other details supplementary tothose set forth herein.

For the purpose of any U.S. national stage filing from this application,all publications and patents mentioned in this disclosure areincorporated herein by reference in their entireties, for the purpose ofdescribing and disclosing the constructs and methodologies described inthose publications, which might be used in connection with the methodsof this disclosure. Any publications and patents discussed above andthroughout the text are provided solely for their disclosure prior tothe filing date of the present application. Nothing herein is to beconstrued as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention.

Unless indicated otherwise, when a range of any type is disclosed orclaimed it is intended to disclose or claim individually each possiblenumber that such a range could reasonably encompass, including anysub-ranges encompassed therein. When describing a range of measurementsevery possible number that such a range could reasonably encompass can,for example, refer to values within the range with one significant digitmore than is present in the end points of a range. Moreover, when arange of values is disclosed or claimed, which Applicants intent toreflect individually each possible number that such a range couldreasonably encompass, Applicants also intend for the disclosure of arange to reflect, and be interchangeable with, disclosing any and allsub-ranges and combinations of sub-ranges encompassed therein.Accordingly, Applicants reserve the right to proviso out or exclude anyindividual members of any such group, including any sub-ranges orcombinations of sub-ranges within the group, if for any reasonApplicants choose to claim less than the full measure of the disclosure.

The invention claimed is:
 1. A cell-free pharmaceutical formulationcomprising: a therapeutically effective amount of exosomes, the exosomesbeing harvested from a heterogeneous cell population that is collectedfrom blood from a donor who has been treated with filagrastim as amobilizing agent, the heterogeneous cell population comprisinghematopoietic cells, hematopoietic stem cell, hematopoietic progenitorcells, T cells, and natural killer cells; and a synthetic antibiotic;wherein the heterogeneous cell population is collected from the donorusing apheresis; wherein the exosomes are harvested from theheterogeneous cell population after exposing the heterogeneous cellpopulation to culture conditions that promote the secretion of exosomes;wherein the culture conditions comprise exosomes-free media; and whereinthe therapeutically effective amount of exosomes is an amount sufficientto increase telomere length.
 2. The pharmaceutical formulation of claim1, further comprising one or more pharmaceutically acceptable carriers,adjuvants, or vehicles.
 3. The formulation of claim 1 further comprisingone or more surfactants, thickening agents, antioxidants, viscositystabilizers, buffers, preservatives, vitamins, antibiotics, or acombination thereof.
 4. The formulation of claim 1 wherein theformulation comprises phosphate buffered saline.
 5. The formulation ofclaim 1, further comprising a vitamin.